A  TEXT-BOOK 

UPON  THE 


PATHOGENIC  BACTERIA 
AND  PROTOZOA 

FOR  STUDENTS  OF  MEDICINE  AND  PHYSICIANS 


BY 

JOSEPH  McFARLAND,  M.  D.,  Sc.  D. 

Professor  of  Pathology  and  Bacteriology  in  the  Medico-Chirurgical  College,  Philadelphia; 

Pathologist  to  the  Philadelphia  General   Hospital  and  to   the   Medico-Chirurgical 

Hospital,  Philadelphia;  Fellow  of  the  College  of  Physicians  of  Philadelphia 


EIGHTH  EDITION,  REVISED 
WITH  323  ILLUSTRATIONS 
A  NUMBER  IN  COLORS 


PHILADELPHIA  AND  LONDON 

W.    B.    SAUNDERS   COMPANY 

1916 


< 


Copyright,  1896.  by  W.  B.  Saunders.  Reprinted  September,  1896.  Re- 
vised, reprinted,  and  recopyrighted  August,  1898.  Reprinted  November, 
1898.  Revised,  reprinted,  and  recopyrighted  August,  1900.  Reprinted 
June,  1901.  Revised,  entirely  reset,  reprinted,  and  recopyrighted  May, 
1903.  Reprinted  August,  1904.  Revised,  reprinted,  and  recopyrighted 
May,  1906.  Reprinted  August,  1907,  and  May,  1908.  Revised,  reprinted, 
and  recopyrighted  August,  1909.  Revised,  reprinted,  and  recopyrighted 
Feptember,  1912.  Reprinted  May,  1914.  Revised,  entirely  reset,  re- 
printed, and  recopyrighted  November,  1915. 


Copyright,  1915,  by  W.  B.  SAUNDERS  COMPANY. 


PRINTED    IN    AMERICA 


PRESS    OF 

B.    SAUNDERS    COMPANY 
PHILADELPHIA 


TO 

MY  HONORED  AND  BELOVED  GRANDFATHER 
/Ifcr.  Jacob 


WHOSE   PARENTAL   LOVE    AND    LIBERALITY    ENABLED    ME    TO    PURSUE 
MY   MEDICAL    EDUCATION 


THIS    BOOK    IS    AFFECTIONATELY    DEDICATED 


PREFACE  TO  THE  EIGHTH  EDITION 


IT  is  a  difficult  thing  to  write  a  preface  for  an  Eighth  Edition. 
No  part  of  the  work  was  found  to  be  so  embarrassing  or  was  sub- 
jected to  greater  procrastination. 

What  can  be  said  that  has  not  been  said  seven  times  already? 
Probably  very  little  about  the  book ;  certainly  very  much  about  the 
feelings  of  the  author. 

He  desires  to  express  to  those  who  have  already  made  acquaintance 
with  the  book,  and  may  with  friendly  feelings  look  into  its  new 
edition,  his  sincere  satisfaction  and  appreciation  of  the  hearty 
receptions  that  have  been  accorded  his  previous  attempts.  He  also 
desires  to  thank  his  reviewers  for  some  helpful  criticisms. 

So  numerous  are  the  additions,  subtractions  and  alterations 
to  which  the  seventh  edition  was  submitted  in  the  preparation  of 
this  Eighth  Edition,  that  it  might  almost  be  said  that  the  text  had 
been  rewritten.  Indeed  they  were  such  that  the  type  of  the  entire 
book  has  been  reset.  It  now  appear  with  slightly  larger  pages; 
in  two  sizes  of  type,  and  gives  a  general  effect  of  contraction,  though 
there  is  really  an  expansion  of  matter  that  would  have  covered  more 
than  fifty  of  the  old-size  pages. 

The  "Pathogenic  Bacteria  and  Protozoa"  is  a  medical  work. 
It  is  hoped  that  it  shall  be  found  helpful  to  medical  workers — 
students  and  practitioners  of  every  class. 

PHILADELPHIA,  PA.  THE  AUTHOR. 

November,  1915. 


CONTENTS 

I  PART  I.— GENERAL 

PAGE 
HISTORICAL  INTRODUCTION I7 

CHAPTER  I 
STRUCTURE  AND  CLASSIFICATION  OF  THE  MICRO-ORGANISMS 26 

CHAPTER  II 
BIOLOGY  OF  MICRO-ORGANISMS 50 

CHAPTER  III 
INFECTION 66 

CHAPTER  IV 
IMMUNITY 88 

CHAPTER  V 
METHODS  OF  OBSERVING  MICRO-ORGANISMS 144 

CHAPTER  VI 
STERILIZATION  AND  DISINFECTION 167 

CHAPTER  VII 

CULTURE-MEDIA  AND  THE  CULTIVATION  OF  MICRO-ORGANISMS 187 

CHAPTER  VIII 
CULTURES,  AND  THEIR  STUDY 201 

CHAPTER  IX 
THE  CULTIVATION  OF  ANAEROBIC  ORGANISMS 215 

CHAPTER  X 
EXPERIMENTATION  UPON  ANIMALS ; 222 

CHAPTER  XI 

THE  IDENTIFICATION  OF  SPECIES 230 

CHAPTER  XII 
THE  BACTERIOLOGY  OF  THE  AIR 234 

CHAPTER  XIII 

THE  BACTERIOLOGY  OF  WATER 237 

CHAPTER  XIV 

THE  BACTERIOLOGY  OF  THE  SOIL 243 

13 


14  Contents 

CHAPTER  XV 

PAGE 
THE  BACTERIOLOGY  OF  FOODS 245 

CHAPTER  XVI 

THE  DETERMINATION  OF  THE  THERMAL  DEATH-POINT  OF  BACTERIA   .    .    249 

CHAPTER  XVII 

THE  DETERMINATION  OF  THE  VALUE  OF  ANTISEPTICS,  GERMICIDES,  AND  DIS- 
INFECTANTS   251 

CHAPTER  XVIII 

BACTERIO- VACCINES 263 

CHAPTER  XIX 
THE  PHAGOCYTIC  POWER  OF  THE  BLOOD  AND  THE  OPSONIC  INDEX  ...    270 

CHAPTER  XX 
THE  WASSERMANN  REACTION  FOR  THE  DIAGNOSIS  OF  SYPHILIS     ....   279 

PART  II.— THE  INFECTIOUS  DISEASES  AND  THE 
SPECIFIC  MICRO-ORGANISMS 

CHAPTER  I 
SUPPURATION 299 

CHAPTER  II 
MALIGNANT  EDEMA 329 

CHAPTER  III 
TETANUS 340 

CHAPTER  IV 
"»     ANTHRAX 352 

CHAPTER  V 
HYDROPHOBIA,  LYSSA,  OR  RABIES 363 

CHAPTER  VI 

ACUTE  ANTERIOR  POLIOMYELITIS 381 

CHAPTER  VII 

CEREBRO-SPINAL  MENINGITIS 386 

CHAPTER  VIII 
GONORRHEA .  394 

CHAPTER  IX 
CATARRHAL  INFLAMMATION 400 

CHAPTER  X 
CHANCROID 403 


I  .  Contents  15 

CHAPTER  XI 

PAGE 
ACUTE  CONTAGIOUS  CONJUNCTIVITIS  ......    f    ..........  406 

CHAPTER  XII 

DIPHTHERIA 411 

CHAPTER  XIII 

VINCENT'S  ANGINA 433 

CHAPTER  XIV 

—  THRUSH 438 

CHAPTER  XV 

WHOOPING-COUGH - 441 

CHAPTER  XVI 

PNEUMONIA 444 

CHAPTER  XVII 

INFLUENZA 462 

CHAPTER  XVIII 

MALTA  AND  MEDITERRANEAN  FEVER 467 

CHAPTER  XIX 

MALARIA 471 

CHAPTER  XX 

RELAPSING  FEVER 494 

CHAPTER  XXI 

SLEEPING  SICKNESS 506 

CHAPTER  XXII 

KALA-AZAR  (BLACK  SICKNESS) 525 

CHAPTER  XXIII 

YELLOW  FEVER 53*6 

CHAPTER  XXIV 

TYPHUS  FEVER 540 

CHAPTER  XXV 

PLAGUE 543 

CHAPTER  XXVI 

ASIATIC  CHOLERA 568 

CHAPTER  XXVII 
TYPHOID  FEVER  .  5^9 


1 6  Contents 

CHAPTER  XXVIII 

PAGE 
DYSENTERY 631 

CHAPTER  XXIX 

: s  TUBERCULOSIS      . 656 

CHAPTER  XXX 

—  LEPROSY 695 

CHAPTER  XXI 

GLANDERS 706 

CHAPTER  XXII 

RHINOSCLEROMA 715 

CHAPTER  XXIII 

— •       SYPHILIS 718 

CHAPTER  XXIV 

FRAMBESIA  TROPICA  (YAWS) 729 

CHAPTER  XXV 

— >      ACTINOMYCOSIS 732 

CHAPTER  XXVI 

MYCETOMA,  OR  MADURA-FOOT .'   .    .   741 

CHAPTER  XXXVII 

_»     BLASTOMYCOSIS •   747 

CHAPTER  XXXVIII 

RINGWORM •    .  • 752 

CHAPTER  XXXIX 

FAVUS    . 755 

CHAPTER  XL 

—  SPOROTRICHOSIS  . 759 


BIBLIOGRAPHIC  INDEX 767 

INDEX  . 7^3 


PART  I.  GENERAL 


HISTORICAL  INTRODUCTION 

BIOLOGY,  chemistry,  medicine,  and  surgery,  in  their  evolution, 
contributed  to  a  new  branch  of  knowledge,  Bacteriology,  whose 
subsequent  development  has  become  of  inestimable  importance  to 
each.  Indeed,  bacteriology  illustrates  the  old  adage,  "The  child 
is  father  of  the  man,"  for  while  it  is  in  part  the  offspring  of  the 
medicine  of  the  past,  it  has  established  itself  as  the  dictator  of  the 
medicine  of  the  present  and  future,  especially  so  far  as  concerns 
the  infectious  diseases. 

THE  EVOLUTION  OF  BACTERIOLOGY 
I.  BIOLOGIC  CONTRIBUTIONS;  THE  DOCTRINE  OF  SPONTANEOUS  GENERATION 

Among  the  early  Greeks  we  find  that  Anaximander  (43d  Olym- 
piad, 6 10  B.  C.)  of  Miletus  held  the  theory  that  animals  were 
formed  from  moisture.  Empedocles  of  Agrigentum  (450  B.  C.) 
attributed  to  spontaneous  generation  all  the  living  beings  which  he 
found  peopling  the  earth.  Aristotle  (384  B.  C.)  is  not  so  general  in 
his  view  of  the  subject,  but  asserts  that  "sometimes  animals  are 
formed  in  putrefying  soil,  sometimes  in  plants,  and  sometimes  in  the 
fluids  of  other  animals." 

Three  centuries  later,  in  his  disquisition  upon  the  Pythagorean 
philosophy,  we  find  Ovid  defending  the  same  doctrine  of  spontaneous 
generation,  while  in  the  Georgics,  Virgil  gives  directions  for  the 
artificial  production  of  bees. 

The  doctrine  of  spontaneous  generation  of  life  was  not  only  current 
among  the  ancients,  but  we  find  it  persisting  through  the  Middle 
Ages,  and  descending  to  our  own  generation.  In  1542,  in  his 
treatise  called  "De  Subtilitate,"  we  find  Cardan  asserting  that 
water  engenders  fishes,  and  that  many  animals  spring  from  fermenta- 
tion. Van  Helmont  gives  special  instructions  for  the  artificial 
production  of  mice,  and  Kircher  in  his  "Mundus  Subterraneus " 
(chapter  "De  Panspermia  Rerum")  describes  and  actually  figures 
certain  animals  which  were  produced  under  his  own  eyes  by  the 
transforming  influence  of  water  on  fragments  of  stems  from  different 
plants.* 

About  1671,  Francesco  Redi  seems  to  have  been  the  first  to 
doubt  that  the  maggots  familiar  in  putrid  meat  arose  de  now: 
*  See  Tyndall:  "Floating  Matter  in  the  Air." 

2  17 


1 8  "••Introduction 

"  Watching  meat  in*  its  'passage  from  freshness  to  decay,  prior  to 
the  appearance  of  maggots,  he  invariably  observed  flies  buzzing 
around  the  meat  and  frequently  alighting  on  it.  The  maggots,  he 
thought,  might  be  the  half-developed  progeny  of  these  flies.  Placing 
fresh  meat  in  a  jar  covered  with  paper,  he  found  that  although  the 
meat  putrefied  in  the  ordinary  way,  it  never  bred  maggots,  while 
meat  in  open  jars  soon  swarmed  with  them.  For  the  paper  he 
substituted  fine  wire  gauze,  through  which  the  odor  of  the  meat 
could  rise.  Over  it  the  flies  buzzed,  and  on  it  they  laid  their  eggs, 
but  the  meshes  being  too  small  to  permit  the  eggs  to  fall  through, 
no  maggots  generated  in  the  meat;  they  were,  on  the  contrary, 
hatched  on  the  gauze.  By  a  series  of  such  experiments  Redi 
destroyed  the  belief  in  the  spontaneous  generation  of  maggots  in 
meat,  and  with  it  many  related  beliefs." 

In  1683  Anthony  van  Leeuwenhoek,  justly  called  the  "Father 
of  microscopy,"  demonstrated  the  continuity  of  arteries  and  veins 
through  intervening  capillaries,  thus  affording  ocular  proof  of 
Harvey's  discovery  of  the  circulation  of  the  blood;  discovered 
bacteria,  seeing  them  first  in  saliva,  discovered  the  rotifers,  and  first 
saw  the  little  globules  in  yeast  which  Latour  and  Schwann  subse- 
quently proved  to  be  plants. 

Leeuwenhoek  involuntarily  reopened  the  old  controversy  about 
spontaneous  generation  by  bringing  forward  a  new  world,  peopled 
by  creatures  of  such  extreme  minuteness  as  to  suggest  not  only  a 
close  relationship  to  the  ultimate  molecules  of  matter,  but  an  easy 
transition  from  them. 

In  succeeding  years  the  development  of  the  compound  microscope 
showed  that  putrescent  infusions,  both  animal  and  vegetable, 
teemed  with  minute  living  organisms. 

Abbe  Lazzaro  Spallanzani  (1777)  filled  flasks  with  organic  in- 
fusions, sealed  their  necks,  and,  after  subjecting  their  contents  to 
the  temperature  of  boiling  water,  placed  them  under  conditions 
favorable  for  the  development  of  life,  without,  however,  being  able 
to  produce  it.  Spallanzani 's  critics,  however,  objected  to  his 
experiment  on  the  ground  that  air  is  essential  to  life,  and  that  in 
his  flasks  the  air  was  excluded  by  the  hermetically  sealed  necks. 

Schulze  (1836)  set  this  objection  aside  by  filling  a  flask  only  half 
full  of  distilled  water,  to  which  animal  and  vegetable  matters  were 
added,  boiling  the  contents  to  destroy  the  vitality  of  any  organisms 
which  might  already  exist  in  them,  then  sucking  daily  into  the 
flask  a  certain  amount  of  air  which  was  passed  through  a  series 
of  bulbs  containing  concentrated  sulphuric  acid,  in  which  it  was 
supposed  that  whatever  germs  of  life  the  air  might  contain  would 
be  destroyed.  This  flask  was  kept  from  May  to  August;  air  was 
passed  through  it  daily,  yet  without  the  development  of  any 
infusorial  life. 

It  must  have  been  a  remarkably  germ-free  atmosphere  in  which 


The  History  of  the  Subject  19 

Schulze  worked,  for,  as  was  shown  by  those  who  repeated  his 
experiment,  under  the  conditions  that  he  regarded  as  certainly 
excluding  all  life,  germs  can  readily  enter  with  the  air. 

In  1838  Ehrenberg  devised  a  system  of  classifying  the  minute 
forms  of  life,  a  part  of  which,  at  least,  is  still  recognized  at  the 
present  time. 

The  term  " infusorial  life"  having  been  used,  it  is  well  to  remark 
that  during  all  the  early  part  of  their  recognized  existence  the 
bacteria  were  regarded  as  animal  organisms  and  classed  among  the 
infusoria. 

Tyndall,  stimulated  by  the  work  of  Pasteur,  conclusively  proved 
that  the  micro-organismal  germs  were  in  the  dust  suspended  in  the 
atmosphere,  and  not  ubiquitous  in  distribution.  His  experiments 
were  very  ingenious  and  are  of  much  interest.  First  preparing 
light  wooden  chambers,  with  a  large  glass  window  in  the  front  and  a 
smaller  window  in  each  side,  he  arranged  a  series  of  test-tubes  in 
the  bottom,  half  in  and  half  out  of  the  chamber,  and  a  pipet,  working 
through  a  rubber  diaphragm,  in  the  top,  so  that  when  desired  the 
tubes,  one  by  one,  could  be  filled  through  it.  Such  chambers  were 
allowed  to  stand  until  all  the  contained  dust  had  settled,  and  then 
submitted  to  an  optical  test  to  determine  the  purity  of  the  contained 
atmosphere  by  passing  a  powerful  ray  of  light  through  the  side 
windows.  When  viewed  through  the  front,  this  ray  was  visible 
only  so  long  as  there  were  particles  suspended  in  the  atmosphere  to 
reflect  it.  When  the  dust  had  completely  settled  and  the  light  ray 
had  become  invisible  because  of  the  purity  of  the  contained  atmos- 
phere, the  tubes  were  cautiously  filled  with  urine,  beef -broth,  and  a 
variety  of  animal  and  vegetable  broths,  great  care  being  taken  that 
in  the  manipulation  the  pipet  should  not  disturb  the  dust.  Their 
contents  were  then  boiled  by  submergence  in  a  pan  of  hot  brine 
placed  beneath  the  chamber,  in  contact  with  the  projecting  ends  of 
the  tubes,  and  subsequently  allowed  to  remain  undisturbed  for 
days,  weeks,  or  months.  In  nearly  every  case  life  failed  to  develop 
in  the  infusions  after  the  purity  of  the  atmosphere  was  established. 

H.  CHEMIC  CONTRIBUTIONS;  FERMENTATION  AND  PUTREFACTION 

As  in  the  world  of  biology  the  generation  of  life  was  an  all- 
absorbing  problem,  so  in  the  world  of  chemistry  the  phenomena  of 
fermentation  and  putrefaction  were  inexplicable  so  long  as  the 
nature  of  the  ferments  was  not  understood. 

In  the  year  1837  La  tour  and  Schwann  succeeded  in  demonstrating 
that  the  minute  oval  bodies  which  had  been  observed  in  yeast  since 
the  time  of  Leeuwenhoek  were  living  organisms — vegetable  forms — 
capable  of  growth. 

So  long  as  yeast  was  looked  upon  as  an  inert  substance  it  was 
impossible  to  understand  how  it  could  impart  fermentation  to  other 
substances;  but  when  it  was  shown  by  Latour  that  the  essential 


(20  Introduction 

element  of  yeast  was  a  growing  plant,  the  phenomenon  became  a 
•^perfectly  natural  consequence  of  life.  Not  only  the  alcoholic,  but 
also  the  acetic,  lactic,  and  butyric  fermentations  have  been  shown 
to  result  from  the  energy  of  low  forms  of  vegetable  life,  chiefly 
bacterial  in  nature.  Prejudice,  however,  prevented  many  chemists 
from  accepting  this  view  of  the  subject,  and  Liebig  strenuously 
adhered  to  his  theory  that  fermentation  was  the  result  of  the 
internal  molecular  movements  which  a  body  in  the  course  of  de- 
composition communicates  to  other  matter  whose  elements  are 
connected  by  a  very  feeble  affinity. 

Pasteur  was  the  first  to  prove  that  fermentation  is  an  ordinary 
chemic  transformation  of  certain  substances,  taking  place  as  the 
result  of  the  action  of  living  cells,  and  that  the  capacity  to  produce  it 
resides  in  all  animal  and  vegetable  cells,  though  in  varying  degree. 

In  1862  he  published  a  paper  "On  the  Organized  Corpuscles 
Existing  in  the  Atmosphere,"  in  which  he  showed  that  many  of  the 
floating  particles  collected  from  the  atmosphere  of  his  laboratory 
were  organized  bodies.  If  these  were  planted  in  sterile  infusions, 
abundant  crops  of  micro-organisms  were  obtained.  By  the  use  of 
more  refined  methods  he  repeated  the  experiments  of  others,  and 
showed  clearly  that  "the  cause  which  communicated  life  to 
his  infusions  came  from  the  air,  but  was  not  evenly  distributed 
through  it." 

Three  years  later  he  showed  that  the  organized  corpuscles  which 
he  had  found  in  the  air  were  the  spores  or  seeds  of  minute  plants, 
and  that  many  of  them  possessed  the  property  of  withstanding  the 
temperature  of  boiling  water — a  property  which  explained  the 
peculiar  results  of  many  previous  experimenters,  who  failed  to 
prevent  the  development  of  life  in  boiled  liquids  inclosed  in  her- 
metically sealed  flasks. 

Chevreul  and  Pasteur,  by  having  proved  that  animal  solids  do  not 
putrefy  or  decompose  if  kept  free  from  the  access  of  germs,  suggested 
to  surgeons  that  putrefaction  in  wounds  is  due  rather  to  the  entrance 
of  something  from  without  than  to  changes  within.  The  deadly 
nature  of  the  discharges  from  putrescent  wounds  had  been  shown  in 
a  rough  manner  by  Gaspard  as  early  as  1822  by  injecting  some  of  the 
material  into  the  veins  of  animals. 

III.  MEDICAL  AND  SURGICAL  CONTRIBUTIONS;  THE  STUDY  OF  THE 
INFECTIOUS  DISEASES 

Probably  the  first  writing  in  which  a  direct  relationship  between 
micro-organisms  and  disease  is  suggested  is  by  Varro,  who  says: 
"It  is  also  to  be  noticed,  if  there  be  any  marshy  places,  that  certain 
minute  animals  breed  [there]  which  are  invisible  to  the  eye,  and  yet, 
getting  into  the  system  through  mouth  and  nostrils,  cause  serious 
disorders  (diseases  which  are  difficult  to  treat)." 

Surgical  methods  of  treatment  depending  for  their  success  upon 


The  History  of  the  Subject  21 

exclusion  of  the  air,  and  of  course,  incidentally  if  unknowingly, 
exclusion  of  bacteria,  seem  to  have  been  practised  quite  early, 
Theodoric,  of  Bologne,  about  1260  taught  that  the  action  of  the  air 
upon  wounds  induced  a  pathologic  condition  predisposing  to  sup- 
puration.  He  also  treated  wounds  with  hot  wine  fomentations. 
The  wine  was  feebly  antiseptic,  kept  the  surface  free  from  bacteria, 
and  the  treatment  was,  in  consequence,  a  modification  of  what  in 
later  centuries  formed  antiseptic  surgery. 

Henri  de  Mondeville  in  1306  went  even  further  than  Theodoric, 
whom  he  followed,  and  taught  the  necessity  of  bringing  the  edges 
of  a  wound  together,  covered  it  with  an  exclusive  plaster  com- 
pounded of  turpentine,  resin,  and  wax,  and  then  applied  the  hot  wine 
fomentation. 

In  1546  Geronimo  Fracastorius  published  at  Venice  a  work 
"De  contagione  et  cont  agio  sis  morbis  et  curatione"  in  which  he  divided 
infectious  diseases  into — 

1.  Those  infecting  by  immediate  contact  (true  contagions). 

2.  Those  infecting  through  intermediate  agents,  such  as  fomites. 

3.  Those  infecting  at  a  distance  or  through  the  air.     He  mentions 
as  belonging  to  this  class  phthisis,  the  pestilential  fevers,  and  a 
certain  kind  of  ophthalmia  (conjunctivitis). 

"  In  his  account  of  the  true  nature  of  disease  germs,  or  seminaria 
contagionum,  ...  he  describes  them  as  particles  too  small  to  be 
apprehended  by  our  senses,  but  as  capable  in  appropriate  media  of 
reproduction,  and  in  this  way  of  infecting  surrounding  tissues. 

"  These  pathogenic  units  Fracastorius  supposed  to  be  of  the 
nature  of  colloidal  systems,  for  if  they  were  not  viscous  or  glutinous 
by  nature  they  could  not  be  transmitted  by  fomites.  Germs 
transmitting  disease  at  a  distance  must  be  able  to  live  in  the  air  a 
certain  length  of  time,  and  this  condition  he  holds  is  possible  only 
when  the  germs  are  gelatinous  or  colloidal  systems,  for  only  hard, 
inert,  discrete  particles  could  endure  longer. 

"  Fracastorius  conceived  that  the  germs  became  pathogenic 
through  the  action  of  animal  heat,  and  in  order  to  produce  disease 
it  is  not  necessary  that  they  should  undergo  dissolution,  but  only 
metabolic  change."* 

In  1671  Kircher  wrote  a  book  in  which  he  expressed  the  opinion 
that  puerperal  fever,  purpura,  measles,  and  various  other  fevers 
were  the  result  of  a  putrefaction  caused  by  worms  or  animalcules. 
His  opinions  were  thought  by  his  contemporaries  to  be  founded 
upon  too  little  evidence,  and  were  not  received. 

Plencig,  of  Vienna,  became  convinced  that  there  was  an  undoubted 
connection  between  the  microscopic  animalcules  exhibited  by  the 
microscope  and  the  origin  of  disease,  and  advanced  this  opinion  as 
early  as  1762. 

In  1704  John  Colbach  described  "a  new  and  secret  method  of 
*  "Brit.  Med.  Jour.,"  May  7,  1910,  p.  1122. 


22  Introduction 

treating  wounds  by  which  healing  took  place  quickly,  without 
inflammation  or  suppuration." 

Boehm  succeeded  in  1838  in  demonstrating  the  occurrence  of 
yeast  plants  in  the  stools  of  cholera,  and  conjectured  that  the 
process  of  fermentation  was  concerned  in  the  causation  of  that 
disease. 

In  1840  Henle  considered  all  the  evidence  that  had  been  collected, 
and  concluded  that  the  cause  of  the  infectious  diseases  was  to  be 
sought  for  in  minute  living  organisms  or  fungi.  He  may  be  looked 
upon  as  the  real  propounder  of  the  GERM  THEORY  OF  DISEASE,  for 
he  not  only  collected  facts  and  expressed  opinions,  but  also  investi- 
gated the  subject  ably.  The  requirements  which  he  formulated  in 
order  that  the  theory  might  be  proved  were  so  severe  that  he  was 
never  able  to  attain  to  them  with  the  crude  methods  at  his  disposal. 
They  were  so  ably  elaborated,  however,  that  in  after  years  they  were 
again  postulated  by  Koch,  and  it  is  only  by  strict  conformity  with 
them  that  the  definite  relationship  between  micro-organisms  and 
disease  has  been  determined. 

Briefly  summarized,  these  requirements  are  as  follows: 

1.  A  specific  micro-organism  must  be  constantly  associated  with 
the  disease. 

2.  It  must  be  isolated  and  studied  apart  from  the  disease. 

3.  When  introduced  into  healthy  animals  it  must  produce  the 
disease,  and  in  the  animal  in  which  the  disease  has  been  experiment- 
ally  produced   the   organism   must   be   found   under   the   original 
conditions. 

In  1843  Dr.  Oliver  Wendell  Holmes  wrote  a  paper  upon  the 
"Contagiousness  of  Puerperal  Fever." 

In  1847  Semmelweiss,  of  Vienna,  struck  by  the  similarity  between 
fatal  wound  infection  with  pyemia  and  puerperal  fever,  cast  aside 
the  popular  theory  that  the  latter  affection  was  caused  by  the 
absorption  into  the  blood  of  milk  from  the  breasts,  and  announced 
his  belief  that  the  disease  depended  upon  poisons  carried  by  the 
fingers  of  physicians  and  students  from  the  dissecting  room  to  the 
woman  in  child-bed,  and  recommended  washing  the  hands  of  the 
accoucheur  with  chlorin  or  chlorid  of  lime,  in  addition  to  the  use 
of  soap  and  water.  He  was  laughed  to  scorn  for  his  pains. 

In  1849  J.  K.  Mitchell,  in  a  brief  work  upon  the  "  Cryptogamous 
Origin  of  Malarious  and  Epidemic  Fevers,"  foreshadowed  the  germ 
theory  of  disease  by  collecting  a  large  amount  of  evidence  to  show 
that  malarial  fevers  were  due  to  infection  by  fungi. 

Pollender  (1849)  and  Davaine  (1850)  succeeded  in  demonstrating 
the  presence  of  the  anthrax  bacillus  in  the  blood  of  animals  suffering 
from  and  dead  of  that  disease.  Several  years  later  (1863)  Davaine, 
having  made  numerous  inoculation  experiments,  demonstrated 
that  this  bacillus  was  the  materies  morbi  of  the  disease.  The  bacillus 
of  anthrax  was  probably  the  first  bacterium  shown  to  be  specific  for  a 


The  History  of  the  Subject  23 

disease.  Being  a  very  large  bacillus  and  a  strongly  vegetative 
organism,  its  growth  was  easily  observed,  while  the  disease  was  one 
readily  communicated  to  animals. 

Klebs,  who  was  one  of  the  pioneers  of  the  germ  theory,  published, 
in  1872,  a  work  upon  septicemia  and  pyemia,  in  which  he  expressed 
himself  convinced  that  the  causes  of  these  diseases  must  come  from 
without  the  body.  Billroth,  however,  strongly  opposed  such  an 
idea,  asserting  that  fungi  had  no  especial  importance  either  in  the 
processes  of  disease  or  in  those  of  decomposition,  but  that,  existing 
everywhere  in  the  air,  they  rapidly  developed  in  the  body  as  soon  as 
through  putrefaction  a  " Faulnisszymoid "  (putrefactive  ferment), 
or  through  inflammation  a  "  Phlogistischezymoid "  (inflammatory 
ferment),  supplying  the  necessary  feeding-grounds,  was  produced. 

In  1873  Obermeier  observed  that  actively  motile,  flexible  spiral 
organisms  were  present  in  large  numbers  in  the  blood  of  patients  in 
the  febrile  stages  of  relapsing  fever. 

In  1875  the  number  of  scientific  men  who  had  entirely  abandoned 
the  doctrine  of  spontaneous  generation  and  embraced  the  germ 
theory  of  disease  was  small,  and  most  of  those  who  accepted  it  were 
experimenters.  A  great  majority  of  medical  men  either  believed, 
like  Billroth,  that  the  presence  of  fungi  where  decomposition  was  in 
progress  was  an  accidental  result  of  their  universal  distribution,  or, 
being  still  more  conservative,  adhered  to  the  old  notion  that  the 
bacteria,  whose  presence  in  putrescent  wounds  as  well  as  in  artificially 
prepared  media  was  unquestionable,  were  spontaneously  generated 
there. 

Before  many  of  the  important  bacteria  had  been  discovered,  and 
while  ideas  upon  the  relation  of  micro-organisms  to  disease  were 
most  crude,  some  practical  measures  were  suggested  that  produced 
greater  agitation  and  incited  more  observation  and  experimentation 
than  anything  suggested  in  surgery  since  the  introduction  of  anes- 
thetics— namely,  antisepsis. 

"It  is  to  one  of  old  Scotia's  sons,  Sir  Joseph  Lister,  that  the 
everlasting  gratitude  of  the  world  is  due  for  the  knowledge  we 
possess  in  regard  to  the  relation  existing  between  micro-organisms 
and  inflammation  and  suppuration,  and  the  power  to  render  wounds 
aseptic  through  the  action  of  germicidal  substances."* 

Lister,  convinced  that  inflammation  and  suppuration  were  due 
to  the  entrance  of  germs  from  the  air,  instruments,  fingers,  etc.,  into 
wounds,  suggested  the  employment  of  carbolic  acid  for  the  purpose 
of  keeping  sterile  the  hands  of  the  operator,  the  skin  of  the  patient, 
the  surface  of  the  wound,  and  the  instruments  used.  He  finally 
concluded  every  operation  by  a  protective  dressing  to  exclude  the 
extrance  of  germs  at  a  subsequent  period. 

Listerism,  or  "antisepsis,"  originated  in  1875,  and  when  Koch 
published  his  famous  work  on  the  "  Wundinf ectionskrankheiten " 
*  Agnew's  "Surgery,"  vol.  i,  chap.  n. 


24  Introduction 

(Traumatic  Infectious  Diseases),  in  1878,  it  spread  slowly  at  first, 
but  surely  in  the  end,  to  all  departments  of  surgery  and  obstetrics. 

From  time  to  time,  as  the  need  for  them  was  realized,  the  genius 
of  investigators  provided  new  devices  which  materially  aided  in  their 
work,  and  have  made  possible  many  discoveries  that  must  otherwise 
have  failed.  Among  them  may  be  mentioned  the  improvement  of 
the  compound  microscope,  the  use  of  sterilized  culture  fluids  by 
Pasteur,  the  introduction  of  solid  culture  media  and  the  isolation 
methods  by  Koch,  the  use  of  the  cotton  plug  by  Schroder  and  van 
Dusch,  and  the  introduction  of  the  anilin  dyes  by  Weigert. 

It  is  interesting  to  note  that  after  the  discovery  of  the  anthrax 
bacillus  by  Pollender  and  Davaine,  in  1849,  there  was  a  period  of 
nearly  twenty-five  years  during  which  no  important  pathogenic 
organisms  were  discovered,  but  during  which  technical  methods  were 
being  elaborated,  making  possible  a  rapid  succession  of  subsequent 
important  discoveries. 

Thus,  in  1873,  Obermeier  discovered  Spirillum  obermeieri  of 
relapsing  fever. 

In  1879  Hansen  announced  the  discovery  of  bacilli  in  the  cells  of 
leprous  nodules,  and  Neisser  discovered  the  gonococcus 

In  1880  the  bacillus  of  typhoid  fever  was  observed  by  Eberth  and 
independently  by  Koch,  Pasteur  published  his  work  upon  "  Chicken- 
cholera,"  and  Sternberg  described  the  pneumococcus,  calling  it 
Micrococcus  pasteuri. 

In  1882  Koch  made  himself  immortal  by  his  discovery  of  and 
work  upon  the  tubercle  bacillus,  and  in  the  same  year  Pasteur 
published  a  work  upon  "  Rouget  du  pore,"  and  Loffler  and  Shiitz 
discovered  the  bacillus  of  glanders. 

In  1884  Koch  reported  the  discovery  of  the  "comma  bacillus," 
the  cause  of  cholera,  and  in  the  same  year  Loffler  isolated  the 
diphtheria  bacillus,  and  Nicolaier  the  tetanus  bacillus. 

In  1892  Canon  and  Pfeiffer  discovered  the  bacillus  of  influenza. 

In  1894  Yersin  and  Kitasato  independently  isolated  the  bacillus 
causing  the  bubonic  plague,  then  prevalent  at  Hong-Kong. 

A  new  era  in  bacteriology,  and  probably  the  most  triumphant 
achievement  of  scientific  medicine,  was  inaugurated  in  1890,  when 
Behring  discovered  the  principles  of  the  "blood-serum  therapy." 
Since  that  time  investigations  have  been  largely  along  the  lines  of 
immunity,  immunization,  and  the  therapeutic  serums,  the  names  of 
Behring,  Kitasato,  Wernicke,  Roux,  Ehrlich,  Metschnikoff,  Bordet, 
Wassermann,  Shiga,  Madsen,  and  Arrhenius  taking  front  rank. 

The  discovery  of  the  Treponema  pallidum,  the  specific  organism 
of  syphilis,  was  made  in  1905  by  Schaudinn  and  Hoffmann,  long 
after  clinical  study  of  the  disease  had  anticipated  it  to  such  an  extent 
that  when  the  discovery  was  finally  made  it  was  unnecessary  to 
modify  our  ideas  of  the  disease  in  any  essential. 


The  History  of  the  Subject  25 

In  the  same  year,  1905,  Castellani  discovered  the  Treponema 
pertenue,  the  cause  of  frambesia  or  yaws. 

In  1911  Noguchi  succeeded  in  obtaining  pure  cultures  of  the 
treponema. 

In  1913  Flexner  and  Noguchi  appear  to  have  been  successful  in 
cultivating  the  virus  of  acute  anterior  poliomyelitis,  in  vitro. 

During  the  time  that  so  much  investigation  of  the  problems  of 
infection  was  in  progress  the  discoveries  were  by  no  means  restricted 
to  the  bacteria  and  their  products,  as  the  reader  might  infer  from 
the  perusal  of  a  chapter  whose  purpose  is  to  explain  the  development 
of  the  department  of  science  now  known  as  Bacteriology.  Other 
organisms  of  different — i.e.,  animal — nature  were  also  found  in  large 
numbers. 

In  1875  Losch  discovered  the  Amoeba  coli;  in  1878  Rivolta  de- 
scribed the  Coccidium  cuniculi  of  the  rabbit;  in  1879  Lewis  first 
saw  Trypanosoma  lewisi  in  the  blood  of  the  rat;  in  1881  Laveran 
discovered  Plasmodium  malarias  in  the  blood  of  cases  of  human 
paludism;  in  1885  Blanchard  described  the  sarcocystis  in  muscle- 
fibers;  in  1893  Councilman  and  Lafleur  studied  Amoeba  dysenteriae 
in  the  stools  and  tissues  of  human  dysentery;  in  1903  Leishman  and 
Donovan  found  the  little  body,  Leishmania  donovani,  in  the  splenic 
juice  of  cases  of  kala-azar,  and  in  1903  Dutton  and  Forde,  working  in- 
dependently, observed  trypanosomes — the  Trypanosoma  gambiense 
of  African  lethargy — in  the  blood  of  human  beings. 

That  the  specific  micro-organisms  of  many  of  the  infectious 
diseases  remained  undiscovered  was  a  source  of  perplexity  so  long  as 
it  was  supposed  that  all  living  things  must  be  visible  to  the  eye  aided 
by  the  microscope.  To-day,  thanks  to  the  invention  of  the  ultra- 
microscope,  that  shows  the  existence  of  things  too  small  to  be 
defined,  and  still  more  to  the  adaptation  of  the  method  of  filtration 
to  the  study  of  the  diseases  in  question,  we  realize  that  the  "viruses" 
of  disease  may  be  visible  or  invisible  and  that  they  have  no  limita- 
tions of  size.  Just  as  bacteria  readily  find  their  way  through  paper 
filters,  so  the  invisible  and  hence  undescribed  viruses — i.e.,  micro- 
organisms— of  yellow  fever,  pleuro-pneumonia  of  cattle,  foot-and- 
mouth  disease,  rinderpest,  hog-cholera,  African  horse-fever,  infec- 
tious anemia  or  swamp  sickness  of  horses,  fowl  plague,  small-pox, 
cow-pox,  sheep-pox,  horse-pox,  swine-pox,  and  goat-pox  are  at  some 
or  all  stages  able  to  pass  through  the  Berkefeld  or  diatomaceous 
earth  filters,  and  some  of  them  through  the  much  less  porous  unglazed 
porcelain  or  Chamberland  filters.  Thus  there  is  opened  a  new 
world  that  is  ultramicroscopic,  but  still  teems  with  invisible  living 
organisms. 


CHAPTER  I 

STRUCTURE    AND    CLASSIFICATION    OF    THE 
MICRO-ORGANISMS 

BACTERIA 

WHEN  Leeuwenhoek  with  his  improved  microscope  discovered 
the  new  world  of  micro-organisms,  he  supposed  them,  on  account 
of  the  active  movements  they  manifested,  to  be  small  animals,  and 
described  them  as  animalculae.  The  early  systematic  writers, 
Ehrenberg  and  Dujardin,  fell  into  the  same  error,  and  it  was  many 
years  before  biologists  had  arrived  at  even  approximate  accuracy  in 
arranging  them.  Indeed,  for  a  long  time  a  great  number  baffled 
systematic  writers,  and  no  less  an  authority  than  Haeckel,  in  1878, 
suggested  that  they  form  a  group  by  themselves  to  be  known  as 
Protista.  Such  a  grouping,  however,  was  unsatisfactory  alike  to 
botanists  and  zoologists,  and,  therefore,  was  used  by  few. 

It  was  evident  that  structure  could  not  be  looked  upon  as  a 
satisfactory  differential  character,  for  between  the  protozoa,  or 
most  simple  animals,  and  the  protophyta,  or  most  simple  plants, 
the  structural  differences  were  too  minute  to  prevent  overlapping. 
Motion  and  locomotion  had  to  be  abandoned,  since  it  was  common 
to  both  groups.  Reproduction  was  likewise  an  unreliable  means 
when  taken  by  itself,  for  much  the  same  means  of.  multiplication 
were  found  to  obtain  in  both  groups.  One  great  physiologic  and 
metabolic  difference  was,  however,  noted:  plants  possess  the  power 
of  nourishing  themselves  upon  purely  inorganic  compounds,  while 
animals  are  unable  to  do  so  and  cannot  live  except  upon  complex 
molecular  combinations  synthesized  by  the  plants.  In  this  metab- 
olic difference  we  find  the  present  criterion  for  the  separation  of  the 
living  organisms  into  the  two  main  groups.  But  this  does  not  dis- 
pose of  all  of  the  difficulties,  for  there  are  certain  small  groups  to 
which  it  does  not  apply.  Thus,  for  example,  the  fungi  which,  when 
judged  by  other  criteria,  are  undoubted  plants,  lack  the  power  of 
inorganic  synthesis,  and  so  resemble  animals. 

Fortunately,  the  question  is  a  purely  academic  one.  Though 
seemingly  at  first  sight  a  most  fundamental  one,  it  is,  in  reality,  of 
trifling  importance,  for  after  a  limited  experience  the  student  un- 
hesitatingly assigns  most  of  the  known  organisms  to  one  or  the 
other  groups,  and  that  occasional  mistakes  may  be  made,  and 
organisms,  like  the  spirochaeta,  appear  sometimes  in  the  group  of 
plants  among  the  bacteria,  and  in  other  writings  in  the  group  of  ani- 
mals among  the  protozoa,  is  a  matter  of  small  consequence  so  long 

26 


Bacteria  27 

as  the  knowledge  of  the  organisms  themselves  is  in  no  particular 
diminished  by  the  method  of  classifying  them. 

In  discussing  the  matter  Delage  says,  "The  question  is  not  so 
important  as  it  appears.  From  one  point  of  view  and  on  purely 
theoretic  grounds  it  does  not  exist,  while  from  another  standpoint 
it  is  insoluble.  If  one  be  asked  to  divide  living  things  into  two 
distinct  groups,  of  which  one  contains  only  animals  and  the  other 
only  plants,  the  question  is  meaningless,  for  plants  and  animals  are 
concepts  which  have  no  objective  reality,  and  in  nature  they  are  only 
individuals.  If  in  considering  those  forms  which  we  regard  as  true 
animals  and  plants  we  look  for  their  phylogenetic  history  and  decide 
to  place  all  of  their  allies  in  one  or  the  other  group,  we  are  sure  to 
reach  no  result;  such  attempts  have  always  been  fruitless." 

"Huxley  pointed  out  as  early  as  1876  the  extremely  close  relation- 
ship between  the  lowest  algae  and  some  of  the  flagellates,  and  it  is 
the  general  opinion  that  no  one  feature  separates  the  lowest  plants 
from  the  lowest  animals,  and  the  difficulty — in  many  cases  the 
impossibility — of  distinguishing  between  them  is  clearly  recognized. 

"The  point  of  view  which  demands  a  strict  separation  of  animals 
and  plants  has,  however,  little  utility  save,  perhaps,  to  determine 
the  limits  of  a  text-book  or  a  monograph."* 

The  relative  position  of  the  pathogenic  vegetable  micro-organisms 
to  the  other  vegetable  organisms  can  be  determined  by  reference  to 
the  following  table.  The  wide  separation  of  the  bacteria  in  Group 
II.  and  all  of  the  others,  which  appear  in  Group  X.,  should  be  noted. 

The  various  genera  to  which  the  pathogenic  fungi  belong  are  by 
no  means  closely  related  to  one  another,  as  can  at  once  be  seen  by  the 
following  amplification  of  Group  X.  Eumycetes: 

No  entirely  satisfactory  grouping  of  the  bacteria  themselves 
has  yet  been  achieved,  the  best  characters  to  be  used  as  the  basis  of 
classification  being  undecided.  The  best  system  for  their  provi-  - 
sional  arrangement  is  probably  that  of  Migula,|  or  the  modification 
of  it  suggested  by  F.  D.  Chester,  J  in  which  the  morphology,  sporula- 
tion,  and  appendages  of  the  bacteria  all  enter  as  important  features. 

Size. — Bacteria  are  so  minute  that  a  special  unit  has  been  adopted 
for  their  measurement.  This  is  the  micron,  micromillimeter  or 
/*,  and  is  the  one-thousandth  part  of  a  millimeter,  equivalent  to  the 
one- twenty-five- thousandth  (J^sooo)  of  an  inch. 

There  is  no  limit  to  the  minuteness  of  micro-organisms.  Visibility 
is  no  longer  a  criterion.  There  are  micro-organisms  that  can  be 
seen  with  low  powers,  others  that  can  only  be  seen  with  high 
powers,  and  a  few  that  probably  cannot  be  seen  with  any  power  of 

*  Calkins',    "The    Protozoa,"    p.    23. 

t"  System  der  Bakterien,"  Jena,  1897-1900  (vols.  i  and  n  appearing  at 
different  times). 

J  "Preliminary  Arrangement  of  the  Species  of  the  Genus  Bacterium,"  "Ninth 
Annual  Report  of  the  Delaware  College  Agricultural  Experiment  Station," 
1897,  Newark,  Delaware,  U.  S.  A. 


28          Structure  and  Classification  of  Micro-organisms 

the  microscope.  These  are  called  "  in  visible  viruses."  They  are 
known  to  us  through  the  biological  quality  of  nitrates  in  which  they 
are  present  because  of  their  ability  to  pass  through  the  pores  of  the 
filters.  For  this  reason  they  are  also  called  "filterable  viruses." 
As  they  cannot  be  seen,  we  have  no  way  of  classifying  them;  they 
may  be  bacteria  or  protozoa,  or  neither  or  both. 


TABLE  I 

THE  PLANT  KINGDOM 


2  3  t?  These  primary  divisions,  like  the  cor- 

p,p  p  responding  primary  division  of  animals 

£  3.5  into  vertebrata  and  invertebrata,   are 

ago  o  now  falling  into  disuse. 


"aHL  p 


?         fc 
I         8 


W    W    w^nONU1^ 

fIRlIf  W  IIIIIH  m 
rwas'i& 


>iyi  i!  tfiF 

Eir^g 

N  N    CL«  .. 


•Mrl'f  s 

0°^?& 
O'S-X-TS 


Bacteria  29 

TABLE  II 

X.  Eumycetes  (eu  good,  JUNTOS  fungus).     The  true  fungi:  plants  without 

chlorophyl. 

Class  i.  Phycomycetes  (</>UKOS  seaweed),  alga-like  fungi. 
Order  i.  Zygomycetes. 

S  ub-order — M  ucorineae . 
Family — Mucoraceae. 
Genus — Mucor. 
Order  2.  Oomycetes. 

Class  2.  Hemiascomycetes. 
Order  i.  Hemiascales. 

Family — Saccharomycetaceae. 
Genus — Saccharomyces. 
"     , — Blastomyces  (?). 
Class  3.  Euascomycetes.  f  Fungi  imperfecti. 

Order  i.  Euascales  (contains  45  families).  I    This  is  a  large  sup- 


Family — Aspergillaceae. 

Genus — Aspergillus. 

"     — Penicillium. 


Class  4.  Laboulbeniomycetes. 
Order  i.  Laboulbeniales. 


plementary  group,  of 
imperfecti}^     known 
fungi  not  included  in 
the  tabulation. 
In  it  we  find  Oidium. 


Class  5.  Basidiomycetes. 
Sub-class — Hemibasidii. 

Order  i.   Hemibasidiales. 

Family — Ustilaginaceae  (smuts). 
Sub-class — Eubasidii. 

Order  i.   Protobasidiomycetes. 

Family — Uredineineae  (rusts). 
Order  2.  Autobasidiomycetes  (mushrooms,  toad-stools,  etc.). 

CLASSIFICATION  OF  THE  BACTERIA 

I.  ORDER:  EUBACTERIA  (True  Bacteria) 

A.  SUB-ORDER:  Haplobacteria  (Lower  Bacteria) 

I.  Family  COCCACE.E.  Cells  globular,  becoming  slightly  elongate  before 
division.  Division  in  one,  two,  or  three  directions  of  space.  Forma- 
tion of  endospores  very  rare. 

(A)  Without  flagella. 

1.  Streptococcus.     Division    in    one    direction    of    space,    producing 

chains  like  strings  of  beads. 

2.  Micrococcus.     Division  in  two  directions  of  space,  so  that  tetrads 

are  often  formed. 

3.  Sarcina.     Division  in   three    directions   of    space,    leading    to    the 

formation  of  bale-like  packages. 

(B)  With  flagella. 

1.  Planococcus.     Division  in  two  directions  of  space,  like  micrococcus. 

2.  Piano sarcina.     Division  in  three  directions,  like  sarcina. 

II.  FamJly  BACTERIACE^;.  Cells  more  or  less  elongate,  cylindric,  and 
straight.  They  never  form  spiral  windings.  Division  in  one  direction 
of  space  only,  transverse  to  the  long  axis  of  the  cell. 

(A)  Without  flagella. 

1.  Bacterium.     Occasional  endospores. 

(B)  With  flagella. 

2.  Bacillus.     Flagella  arising  from  any   part  of  the  surface.     Endo- 

spore-formation  common. 

3.  Pseudomonas.     Flagella  attached  only   at    the    ends    of    the    cell. 

Endospores  very  rare. 

III.  Family  SPIRILLACE^E.  Cells  twisted  spirally  like  a  corkscrew,  or 
representing  sections  of  the  spiral.  Division  only  transverse  to  the 
long  diameter. 


30          Structure  and  Classification  of  Micro-organisms 

1.  Spirosoma.     Rigid;  without  flagella. 

2.  Microspira.     Rigid;  having  one,  two,  or  three  undulating  flagella 

at  the  ends. 

3.  Spirillum.     Rigid;  having  from  five  to  twenty  curved  or  undulat- 

ing flagella  at  the  ends. 

4.  Spiroch&ta.*     Serpentine    and    flexible.      Flagella    not    observed; 

probably  swim  by  means  of  an  undulating  membrane. 

B.  SUB-ORDER:    Trichobacteria    (Higher    Bacteria) 

IV.  Family  MYCOBACTERIACE^.  Cells  forming  long  or  short  cylindric 
filaments,  often  clavate-cuneate  or  irregular  in  form,  and  at  times 
showing  true  or  false  branchings.  No  endospores,  but  formation  of 
gonidia-like  bodies  due  to  segmentation  of  the  cells.  No  flagella. 
Division  at  right  angles  to  the  axis  of  rod  in  filament.  Filaments  not 
surrounded  by  a  sheath  as  in  Chlamydobacteriaceae. 

1.  Mycobacterium.     Cells    in    their    ordinary    form,    short    cylindric 

rods  often  bent  and  irregularly  cuneate.  At  times  Y-shaped 
forms  or  longer  filaments  with  true  branchings  may  produce 
short  coccoid  elements,  perhaps  gonidia.  (This  genus  includes 
the  Corynebacterium  of  Lehmann-Neumann.)  No  flagella. 

2.  Aclinomyces.     Cells  in  their  ordinary  form  as  long  branched  fila- 

ments; growth  coherent,  dry  or  crumpled.  Produce  gonidia- 
like  bodies.  Cultures  generally  have  a  moldy  appearance,  due 
to  the  development  of  aerial  hyphae.  No  flagella. 

V.  Family  CHLAMYDOBACTERIACEAE.  Forms  that  vary  in  different  stages 
of  their  development,  but  all  characterized  by  a  surrounding  sheath 
about  both  branched  and  unbranched  threads.  Division  transverse 
to  the  length  of  the  filaments. 

1.  Cladothrix.     Characterized    by    pseudo-dichotomous    branchings. 

Division  only  transverse.  Multiplication  by  the  separation  of 
whole  branches.  Transplantation  by  means  of  polar  flagellated 
swarm-spores. 

2.  Crenothrix.     Cells  united  to  form  unbranched  threads  which  in 

the  beginning  divide  transversely.  Later  the  cells  divide  in  all 
three  directions  of  space.  The  products  of  final  division  become 
spheric,  and  serve  as  reproductive  elements. 

3.  Phragmidiothrix.     Cells  at  first  united  into  unbranched  threads. 

Divide  in  three  directions  of  space.  Late  in  the  development, 
by  the  growth  of  certain  of  the  cells  through  the  delicate,  closely 
approximated  sheath,  branched  forms  are  produced. 

4.  Thiothrix.     Unbranched  cells  inclosed  in  a  delicate  sheath.     Non- 

motile.  Division  in  one  direction  of  space.  Cells  contain  sulphur 
grains. 

II.  ORDER:  THIOBACTERIA  (Sulphur  Bacteria) 

I.  Family  BEGGIATOACE^E.  Cells  united  to  form  threads  which  are  not 
surrounded  by  an  inclosing  sheath.  The  septa  are  scarcely  visible. 
Divide  in  one  direction  of  space  only.  Motility  accomplished  through 
the  presence  of  an  undulating  membrane.  Cells  contain  sulphur 
grains. 
There  are  two  families,  numerous  sub-families,  and  thirteen  genera  in  this 

order.     They  are  all  micro-organisms  of  the  water  and  soil,  and  have  no 

interest  for  the  medical  student. 

Structure. — Nucleus. — When  subjected  to  the  action  of  nuclear 
stains,  large  vague  nuclear  formations  are  usually  observed  in  the 
bacterial  cells,  f 

*  The  spirochseta  and  some  closely  related  forms  are  now  thought  to  be 
more  properly  classified  among  the  protozoa  than  among  the  bacteria.  They 
will,  therefore,  appear  again  in  the  tabulation  of  the  protozoan  organisms. 

t  For  literature  upon  the  nucleus  of  the  bacteria,  see  the  lengthy  paper  by 
Douglas  and  Distaso  ("Centralbl.  fur  Bakt.,"  etc.,  I.  Abt.  Orig.,  LXVI,  p.  321). 


Bacteria  31 

Cytoplasm. — The  cytoplasm,  of  which  very  little  exists  between  the 
large  nucleus  and  cell-wall,  is  sometimes  granular,  as  in  Bacillus 
megatherium,  and  sometimes  contains  fine  granules  of  chlorophyl, 
sulphur,  fat,  or  pigment. 

Capsule. — Each  cell  is  surrounded  by  a  distinct  cell- wall,  which  in 
some  species  shows  the  cellulose  reaction  with  iodin. 

The  cell-walls  of  certain  bacteria  at  times  undergo  a  peculiar 
gelatinous  change  or  permit  the  exudation  of  gelatinous  material 
from  the  cytoplasm,  and  appear  surrounded  by  a  halo  or  capsule. 
Such  capsules  are  seen  about  the  pneumococcus  as  found  in  blood 
or  sputum,  Friedlander's  bacillus,  as  seen  in  sputum,  Bacillus 
aerogenes  capsulatus  in  blood  or  tissue,  and  many  other  organisms. 
Friedlander  pointed  out  that  the  capsule  of  his  pneumonia  bacillus, 
as  found  in  the  lung  tissue  or  in  the  " prune-juice"  sputum,  was  very 
distinct,  though  it  could  not  be  demonstrated  at  all  when  the  organ- 
isms grew  in  gelatin. 

Polar  Granules. — By  carefully  staining  an  appropriate  organism, 
certain  peculiarities  of  structure  can  sometimes  be  shown.  Thus, 
some  bacilli  contain  distinct  "polar  granules"  (metachromatic  or 
Babes-Ernst  granules) — rounded  or  oval  bodies — situated  at  the 
ends  of  the  cell.  Their  significance  is  unknown.  They  have  been 
supposed  to  bear  some  relationship  to  the  biologic  activity  of  the 
organism,  especially  its  pathogenesis,  but  this  is  uncertain,  and 
Gauss*  and  Schumburgf  believe  that  they  vary  with  the  reaction 
of  the  culture-media  upon  which  the  bacteria  grow  and  have 
nothing  to  do  with  virulence.  The  diphtheria  bacillus  and  the 
cholera  spirillum  stain  very  irregularly  in  fresh  cultures,  as  if 
the  tingeable  substance  were  not  uniformly  distributed  throughout 
the  cytoplasm.  Vacuolated  bacteria  and  bacteria  that  will  not 
stain,  or  stain  very  irregularly,  may  usually  be  regarded  as  degener- 
ated organisms  (involution  forms)  which,  because  of  plasmolysis,  or 
solution,  can  no  longer  stain  uniformly. 

Flagella. — Many  bacteria  possess  delicate  straight  or  wavy 
filaments,  called  flagella,  which  appear  to  be  organs  of  locomotion. 

MesseaJ  has  suggested  that  the  bacteria  be  classified,  according  to 
the  arrangement  of  the  flagella,  into: 

I.  Gymnobacteria  (forms  without  flagella). 
II.  Trichobacteria  (forms  with  flagella). 

1.  Monotricha  (with  a  single  flagellum  at  one  end). 

2.  Lophotricha  (with  a  bundle  of  flagella  at  one  end). 

3.  Amphitricha  (with  a  flagellum  at  each  end). 

4.  Peritricha  (flagella  around  the  body,  springing  from  all  parts  of  its 

surface). 

*  "Centralbl.  f.  Bakt.,"  etc.,  Feb.  5,  1902,  xxxi,  No.  3,  p.  106. 
t  Ibid.,  June  3,  1902,  xxxi,  No.  14,  p.  694. 
t  "Rivista  d'igiene  e  sanata  publica,"  1890,  11. 


32  Structure  and  Classification  of  Micro-organisms 

This  arrangement  is,  however,  less  satisfactory  than  that  of 
Migula  already  given. 

Motility. — The  greater  number  of  the  bacteria  supplied  with 
flagella  are  actively  motile,  the  locomotory  power  no  doubt  being 
the  lashing  flagella.  The  rod  and  spiral  micro-organisms  are  most 
plentifully  supplied  with  flagella;  only  a  few  of  the  spheric  forms  have 
them. 

The  presence  of  flagella,  however,  does  not  invariably  imply 
motility,  as  they  may  also  serve  to  stimulate  the  passage  of  currents 
of  nutrient  fluid  past  the  organism,  and  so  favor  its  nutrition.  The 
flagellate  bacteria  are  more  numerous  among  the  saprophytic  than 
the  pathogenic  forms. 

Bacillus  megatherium  has  a  distinct  but  limited  ameboid  move- 
ment. 

The  dancing  movement  of  some  of  the  spheric  bacteria  seems  to  be  the  well- 
known  Brownian  movement,  which  is  a  physical  phenomenon.  It  is  some- 
times difficult  to  determine  whether  an  organism  viewed  under  the  microscope 
is  really  motile  or  whether  it  is  only  vibrating.  One  can  usually  determine 
by  observing  that  in  the  latter  case  it  does  not  change  its  relative  position  to 
surrounding  objects. 

In  some  cases  the  colonies  of  actively  motile  bacteria,  such  as  the 
proteus  bacilli,  show  definite  migratory  tendencies  upon  5  per  cent, 
gelatin.  The  active  movement  of  the  bacteria  composing  the 
colony  causes  its  shape  constantly  to  change,  so  that  it  bears  a 
faint  resemblance  to  an  ameba,  and  moves  about  from  place  to 
place  upon  the  surface  of  the  gelatin. 

Reproduction. — Fission. — Bacteria  multiply  by  binary  division 
(fission).  A  bacterium  about  to  divide  appears  larger  than  normal, 
and,  if  a  spheric  organism,  more  or  less  ovoid.  By  appropriate 
staining  karyokinetic  changes  may  be  observed  in  the  nuclei. 
When  the  conditions  of  nutrition  are  good,  fission  progresses  with 
astonishing  rapidity.  Buchner  and  others  have  determined  the 
length  of  a  generation  to  be  from  fifteen  to  forty  minutes. 

The  results  of  binary  division,  if  rapidly  repeated,  are  almost 
appalling.  "Cohn  calculated  that  a  single  germ  could  produce  by 
simple  fission  two  of  its  kind  in  an  hour;  in  the  second  hour  these 
would  be  multiplied  to  four,  and  in  three  days  they  would,  if  their 
surroundings  were  ideally  favorable,  form  a  mass  which  can  scarcely 
be  reckoned  in  numbers."  "Fortunately  for  us,"  says  Woodhead, 
"they  can  seldom  get  food  enough  to  carry  on  this  appalling  rate  of 
development,  and  a  great  number  die  both  for  want  of  food  and 
because  of  the  presence  of  other  conditions  unfavorable  to  their 
existence." 

Sporulation. — When  the  conditions  for  rapid  multiplication  by 
fission  are  no  longer  good,  many  of  the  organisms  guard  against 
extinction  by  the  formation  of  spores. 

Endospores,  or  spores  developed  within  the  cells,  are  generally 


Bacteria  33 

formed  in  the  elongated  bacteria — bacillus  and  spirillum — but  Zopf 
has  observed  similar  bodies  in  micrococci.  Escherich  also  claims 
to  have  found  undoubted  spores  in  a  sarcina. 

Spores  may  be  either  round  or  oval.  As  a  rule,  each  organism 
produces  a  single  spore,  which  is  situated  either  at  its  center  or  at  its 
end.  When,  as  sometimes  happens,  the  diameter  of  the  spore  is 
greater  than  that  of  the  bacillus,  it  causes  a  peculiar  barrel  shape 
bulging  of  the  organism,  described  as  clostridium.  When  the  dis- 
tending spore  is  at  the  end,  a  "Trommelschlager,"  or  "  drum- 
stick," is  formed.  End-spores  are  almost  characteristic  of  anaerobic 
bacilli.  When  the  formation  of  a  spore  is  about  to  commence,  a 
small  bright  point  appears  in  the  cytoplasm,  and  increases  in  size 
until  its  diameter  is  nearly  or  quite  as  great  as  that  of  the  bacterium. 
A  dark,  highly  refracting  capsule  is  finally  formed  about  it.  As  soon 
as  the  spore  arrives  at  perfection  the  bacterium  seems  to  die,  as  if  its 
vitality  were  exhausted. 

The  spores  differ  from  the  bacteria  in  that  their  capsules  prevent 
evaporation  and  enable  them  to  withstand  drying  and  the  applica- 
tion of  a  considerable  degree  of  heat.  Very  few  adult  bacteria  are 
able  to  resist  temperatures  above  7o°C.  Spores  are,  however, 

a  b  c  d  e  f 

O  o 


Fig.  i. — Diagram  illustrating  sporulation:  a,  Bacillus  inclosing  a  small  oval 
spore;  b,  drumstick  bacillus,  with  the  spore  at  the  end;  c,  clostridium;  d,  free 
spores;  e  and  /,  bacilli  escaping  from  spores. 

uninjured  by  such  temperatures,  and  can  even  successfully  resist 
the  temperature  of  boiling  water  (ioo°C.)  for  a  short  time.  The 
extreme  desiccation  caused  by  a  protracted  exposure  to  a  dry 
temperature  of  i5o°C.  will  invariably  destroy  them,  as  will 
also  steam  under  pressure.  Not  only  can  the  spores  successfully 
resist  a  considerable  degree  of  heat,  but  they  are  also  unaffected  by 
cold  of  almost  any  intensity.  Von  Szekely*  found  anthrax 
spores  capable  of  germination  after  eighteen  years  and  six  months 
in  some  dried-up  old  gelatin  cultures  found  in  his  laboratory. 

Arthrospores. — The  formation  of  arthrospores  is  less  clear,  and 
seems  to  be  the  conversion  of  the  entire  organism  into  a  spore  or 
permanent  form.  Arthrospores  have  been  observed  particularly 
among  the  micrococci,  where  certain  individuals  become  enlarged 
beyond  the  normal,  and  surrounded  by  a  capsule. 

Though  the  cell-wall  of  the  adult  bacterium  is  easily  penetrated 
by  solutions  of  the  anilin  dyes,  it  is  difficult  to  stain  spores,  which  are 
distinctly  more  resistant  to  the  action  of  chemic  agents  than  the 
bacteria  themselves. 

*  "Zeitschr.  fur  Hygiene,"  1903,  XLIV,  3. 


34  Structure  and  Classification  of  Micro-organisms 

Germination  of  Spores. — When  a  spore  is  about  to  germinate,  the 
contents,  which  have  been  clear  and  transparent,  become  granular, 
the  body  increases  slightly  in  size,  the  capsule  becomes  less  distinct, 
and  in  the  course  of  time  splits  open  to  allow  the  escape  of  a  young 
organism.  The  direction  in  which  the  capsule  ruptures  varies  in 
different  species.  Bacillus  subtilis  escapes  from  the  side  of  the 
spore;  Bacillus  anthracis  from  the  end.  This  difference  can  be 
made  use  of  as  an  aid  in  differentiating  otherwise  similar  organisms. 

So  soon  as  the  young  bacillus  escapes  it  begins  to  increase  in  size, 
develops  a  characteristic  capsule,  and  presently  begins  the  propaga- 
tion of  its  species  by  fission. 

Morphology. — The  three  principal  forms  of  bacteria  are  spheres 
(cocci),  rods  (bacilli),  and  screws  (spirilla). 

Cocci. — The  spheric  bacteria,  from  a  fancied  resemblance  to 
little  berries,  are  called  cocci  or  micrococci.  When  they  divide, 
and  the  resulting  organisms  remain  attached  to  one  another,  a 

/ 


g 


Fig.  2. — Diagram  illustrating  the  morphology  of  the  cocci:  a,  Coccus  or 
micrococcus;  b,  diplococcus;  c,  d,  streptococci;  e,  f,  tetracocci  or  merismopedia; 
g,  ht  modes  of  division  of  cocci;  i,  sarcina;  j,  coccus  with  flagella;  k,  staphylococci. 

diplococcus  is  produced.  Diplococci  may  consist  of  two  attached 
spheres,  though  each  half  commonly  shows  flattening  of  the  con- 
tiguous surfaces.  In  a  few  cases,  as  the  gonococcus,  the  approxi- 
mated surfaces  may  be  slightly  concave,  causing  the  organism  to 
resemble  the  German  biscuit  called  a  "Semmel."  When  a  second 
binary  division  occurs,  and  four  resulting  individuals  remain  at- 
tached to  one  another,  without  disturbing  the  arrangement  of  the 
first  two,  a  tetrad,  or  tetracoccus,  is  formed.  To  the  entire  groups 
of  cocci  dividing  in  two  directions  of  space  so  as  to  produce  fours, 
eights,  twelves,  etc.,  on  the  same  plane,  the  name  merismopedia  has 
been  given.  Migula  uses  the  term  micrococcus  for  the  unflagellated 
tetrads,  and  planococcus  for  the  flagellated  forms. 

If  division  takes  place  in  three  directions  of  space,  so  as  to  pro- 
duce a  cubic  "package"  of  cocci,  the  resulting  aggregation  is 
described  as  a  sarcina.  This  form  resembles  a  dice  or  a  miniature 
bale  of  cotton.  Few  sarcinae  have  flagella,  similar  flagellated 
organisms  being  called  by  Migula  planosarcina. 

If  division  always  take  place  in  the  same  direction,  so  that  the 


Bacteria  35 

cocci  remain  attached  to  one  another  like  a  string  of  beads,  the 
organism  is  described  as  a  streptococcus. 

Cocci  commonly  occur  in  irregular  groups  having  a  fancied  re- 
semblance to  bunches  of  grapes.  Such  are  called  staphylococci,  and 
most  organisms  not  finding  a  place  in  the  varieties  already  described 
are  so  classed. 

Cocci  associated  in  globular  or  lobulated  clusters,  incased  in  a 
resisting  gelatinous,  homogeneous  mass  have  been  described  by 
Billroth  as  ascococcus.  Cocci  solitary  or  in  chains,  surrounded  by  an 
incasement  of  almost  cartilaginous  consistence,  have  been  called 
leuconostoc. 


Fig.  3. — Diagram  illustrating  the  morphology  of  the  bacilli:  a,  b,  c,  Various 
forms  of  bacilli;  d,  e,  bacilli  with  flagella;/,  chain  of  bacilli,  individuals  distinct; 
g,  chain  of  bacilli,  individuals  not  separated. 

Bacilli. — Better  known,  if  not  more  important,  bacteria  consist  of 
elongate  or  "rod-shaped  forms,"  and  bear  the  name  bacillus  (a  rod). 
These  present  considerable  variation  of  form.  Some  are  ellipsoid, 
some  long  and  slender.  Some  have  rounded  ends,  as  Bacillus 
subtilis;  others  have  square  ends,  as  B,  anthracis.  Some  are  large, 
some  exceedingly  small.  Some  always  occur  singly,  never  uniting 
to  form  threads  or  chains;  others  are  nearly  always  so  conjoined. 

The  bacilli  divide  by  transverse  fission  only,  so  that  the  only 
peculiarity  of  arrangement  is  the  formation  of  threads  or  chains. 
In  the  older  writings,  short,  stout  bacilli  were  described  under  the 
generic  term  bacterium.  Migula  now  employs  the  term  to  include 
only  bacillary  forms  without  flagella.  A  pseudomonas  is  a  bacillary 


Fig.  4.— Diagram  illustrating  the  morphology  of  the  spirilla:  a,  6,  c,  Spirilla. 

form  with  polar  flagella.  Some  of  the  flexile  bacilli  have  sinuous 
movements  resembling  the  swimming  of  a  snake  or  an  eel,  and  are 
sometimes  described  as  vibrio;  but  this  name  also  has  passed  into 
disuse,  except  in  France. 

Spirilla. — If  a  rod-shaped  bacterium  is  spirally  twisted  and  re- 
sembles a  corkscrew,  it  is  called  spirillum.  The  rigid  forms  without 
flagella  are  known  as  spirosoma;  rigid  forms  with  flagella,  spirilla 
and  microspira. 


36          Structure  and  Classification  of  Micro-organisms 

A  spiral  organism  of  ribbon  shape  is  called  spiromonas,  while  a 
similar  oganism  of  spindle  shape  is  called  a  spirulina.  One  species 
of  spiral  bacteria  in  whose  cytoplasm  sulphur  granules  have  been 
detected  has  been  called  ophidiomonas. 

Spiral  organisms  with  undulating  membranes  are  known  as 
spiroch&ta,  but  these  and  the  similar  genus  treponema  are  now 
regarded  as  more  correctly  placed  among  the  protozoan  organisms. 


THE  HIGHER  BACTERIA 

The  Higher  Bacteria  form  a  group  intermediate  between  the 
Schizomycetes,  or  true  bacteria,  and  the  Hyphomycetes,  or  molds. 
In  the  classification  of  Migula  and  Chester  they  include  the  Myco- 


Fig.    5. — Cladothrix,   showing   false    branching.     (From    Hiss    and    Zinsser, 
"Text-Book    of    Bacteriology,"    D.    Appleton    &    Co.,    publishers.) 

bacteriaceae  and  the  Chlamydobacteriaceae.  Some,  like  Petruschky, 
believe  them  to  be  more  closely  related  to  the  true  molds  than  to  the 
bacteria.  They  are  characterized  by  filamentous  forms  with  real  or 
apparent  branchings.  The  filaments  are  usually  regularly  divided 
transversely,  so  as  to  appear  as  if  composed  of  bacilli.  The  free 
ends  only  seem  to  be  endowed  with  reproductive  functions,  and 
develop  peculiar  elements  that  differentiate  the  higher  from  the 
other  bacteria  whose  cells  are  all  equally  free  and  independent. 

Leptothrix. — These  comprise  long  threads  which  do  not  branch. 
They  are  not  always  easily  separated  from  chains  of  bacilli.  They 
rarely  appear  to  play  a  pathogenic  role,  though  those  inhabiting 
the  mouth  occasionally  secure  a  foothold  upon  the  edges  of  the 
tonsillar- crypts,  where  they  grow,  with  the  formation  of  persistent 
white  patches.  This  form  of  leptothrix  mycosis  is  chronic  and  diffi- 


The  Higher  Bacteria 


37 


cult  to  treat.  The  leptothrix  is  a  very  difficult  organism  to  secure 
in  culture.  The  attempts  of  Vigna)*  and  of  Arustamoff1"  were 
successful,  but  upon  the  usual  culture-media  the  organisms  grew 
very  sparingly. 

Cladothrix. — These  also  produce  long  thread-like  filaments,  but 
they  occasionally  show  what  is  described  as  false  branching;  that  is, 
branches  seem  to  originate  from  the  threads,  but  no  distinct  connec- 
tion between  the  thread  and  the  apparent  branch  obtains.  None  of 
the  cladothrices  is  known  to  be  pathogenic.  They  are  frequent 
organisms  of  the  atmospheric  dust,  and  not  infrequently  appear  as 
"weeds"  in  culture-media.  The  colonies  grow  to  about  a  centi- 
meter in  diameter,  are  usually  white  in  color,  irregularly  rounded, 


Fig.    6. — Streptothrix   enteola.     Film   preparation  from   peptone-beef-broth 
culture,  fourteen  days  at  37°C.  X  1000.     (Foulerton.) 

sharp  at  the  edges,  more  or  less  concentric,  dry  and  powdery  (not 
velvety)  or  scaly  on  the  surface.  They  commonly  liquefy  gelatin 
and  blood- serum. 

Streptothrix. — These  organisms  certainly  branch.  They  also  form 
endospores.  Many  of  them  can  be  cultivated.  Not  a  few  are 
found  under  circumstances  suggesting  pathogenic  action.  For  a  long 
time  there  has  been  a  disposition  to  regard  Bacillus  tuberculosis  as  a 
form  of  Streptothrix,  since  old  cultures  show  branching  involution 
forms.  The  old  genus  actinomyces  is  also  included  by  a  number  of 
writers  among  the  streptothrices,  so  that  the  Actinomyces  bovis  of 
Bellinger  is  called  Streptothrix  actinomyces,  the  Actinomyces 
madurae,  Streptothrix  madurae,  and  the  organism  found  by  Nocard 
in  the  disease  known  as  "farcin.du  bceuf,"  Streptothrix  farcinica. 

*"Annales  de  physiologie,"  1886. 

t"Kolle  and  Wassermann,  "Handbuch  der  Pathogenen  Mikroorganismen," 
1903,  IT,  p.  851;  Wratsch,  1889. 


38  Structure  and  Classification  of  Micro-organisms 

There  seems,  however,  no  adequate  ground  for  this  arrangement, 
and  the  old  genus  Actinomyces  should  be  kept.  Eppinger  found  a 
streptothrix  in  the  pus  of  a  cerebral  abscess,  and  Petruschky, 
Berestneff,  Flexner,  Norris,  and  Larkin  have  found  streptothrices  in 
cases  of  pulmonary  disease  simulating  tuberculosis.  The  organisms 
described  by  these  writers  were  not  identical,  so  that  there  are  prob- 
ably several  different  species.  They  usually  grow  well  upon 
ordinary  media  and  upon  solid  media  form  whitish,  glistening,  well- 
circumscribed  colonies  attaining  a  diameter  of  several  millimeters. 
As  they  grow  old  they  turn  yellowish  or  brownish.  They  liquefy 
gelatin.  Some  of  the  cultures  were  not  harmful  to  the  laboratory 
animals,  others  caused  suppuration. 

Actinomyces. — The  chief  characterization  of  the  organisms  of  this 
group  is  a  clavate  expansion  of  the  terminal  ends  of  radiating  fila- 
ments. These  are  seen  in  sections  of  diseased  tissues  containing  the 
organisms,  but  rarely  are  well  shown  in  the  artificial  cultures.  For 
further  particulars  of  these  organisms  see  Actinomyces  bovis,  etc. 


THE  YEASTS,  OR  BLASTOMYCETES 

The  organisms  of  this  group  are  sharply  separated  from  the 
bacteria  by  their  larger  size,  elliptic  form,  and  by  multiplication  by 
gemmation  or  budding. 


FjK  7  — Blastomycetes  dermatitidis.  .Budding  forms  and  mycelial  growth 
from  glucose  agar.  (Irons  and  Graham,  in  "Journal  of  .Infectious  Diseases  .) 

Each  organism  is  surrounded  by  a  sharply  defined,  doubly 
contoured,  highly  refracting,  transparent  cellulose  envelope.  Com- 
monly each  cell  contains  one  or  more  distinct  vacuoles.  When 
multiplication  is  in  progress,  smaller  and  larger  buds  are  formed. 


The  Oidia  39 

The  yeasts,  of  which  Saccharomyces  cerevisiae  may  be  taken  as 
the  type,  are  active  fermentative  organisms,  quickly  splitting  the 
sugars  into  CO2  and  alcohol,  and  are  largely  cultivated  and  used 
in  the  manufacture  of  fermented  liquors  and  bread.  They  grow  well 
in  fermentable  culture-media  and  most  of  them  also  grow  upon  the 
ordinary  laboratory  culture-media.  Many  varieties,  some  of 
which  produce  red  or  black  pigment,  some  no  pigment  at  all,  are 
known.  They  play  very  little  part  in  the  pathogenic  processes. 
Burse  has  observed  a  case  of  generalized  fatal  infection  caused  by  a» 
yeast  that  he  calls  Saccharomyces  hominis.  Gilchrist,  Curtis, 
Ophiils,  and  others  have  seen  localized  human  infections  by  blasto- 
mycetes.  (See  Blastomycetic  dermatitis.) 

THE  OIDIA 

These  organisms  seem  to  occupy  a  place  intermediate  between  the 
yeasts  and  the  molds — the  blastomycetes  and  the  hyphomycetes. 
In  certain  stages  they  appear  as  oval  cells  which  multiply  by  gem- 


Fig.    8. — Oidium,  showing  the  various  vegetative  and  reproductive  elements. 

X  350-     (Grawitz.) 

mation,  but  instead  of  becoming  separated,  hang  together.  At  a 
later  stage  of  development  they  grow  into  long  filamentous  forma- 
tions suggesting  the  mycelia  of  molds,  but  being  less  regular. 
Certain  cells  also  develop  as  reproductive  organs. 

They  are  common  micro-organisms  of  the  air  and  appear  as 
frequent  causes  of  contamination  in  culture-media,  upon  all  forms 
of  which  they  grow  readily,  producing  liquefaction  where  possible. 
They  engage  in  but  few  pathogenic  processes,  the  most  familiar 
being  that  brought  about  by  Oidium  albicans,  which  causes  the 
common  disease  of  childhood  known  as  thrush  (q.  v.). 


40          Structure  and  Classification  of  Micro-organisms 


THE  MOLDS 


In  this  group  it  is  customary  to  place  a  miscellaneous  collection 
of  organisms  having  in  common  the  formation  of  a  well-marked 


Fig.    9. — Oidium.     (Kolle  and  Wassermann.) 

mycelium,  but  being  so  diversified  in  other  respects  as  to  place  them 
in  widely  separated  groups  in  the  systematic  arrangement  of  the 


Fig.  10. — Mucor  mucedo:  i,  A  sporangium  in  optical  longitudinal  section; 
c,  columella;  m,  wall  of  sporangium;  sp,  spores;  2,  a  ruptured  sporangium  with 
only  the  columella  (c)  and  a  small  portion  of  the  wall  (m)  remaining;  3,  two 
smaller  sporangia  with  only  a  few  spores  and  no  columella;  4,  germinating 
spores;  5,  ruptured  sporangium  of  Mucor  mucilaginus  with  deliquescing  wall 
(m)  and  swollen  interstitial  substance  (z);  sp,  spores.  (After  Brefeld.) 

fungi.     Some  are  correctly  placed  among  the  "Imperfect  fungi," 
some  among  the  Ascomycetes,   and  some  among  the  Phycomy- 


The  Molds  41 

cetes.     They    are    all    active    enzymic    agents    and    produce    fer- 
mentative and  putrefactive  changes. 

1.  Achorion. — The  organisms  of  this  genus  are  characterized  by 
a  more  or  less  branched  hypha,  3  to  5/z  in  diameter,  which  break  up 
after   a    time    into    rounded    or    cuboidal    spores.     The    Achorion 
schonleini  is  higrA^thogenic  and  will  be  described  in  the  section 
upon  Favus. 

2.  Tricophyton  and  Microsporon. — These  names  are  applied  some- 
what loosely  to  organisms  affecting  skin  and  hair  follicles  of  men  and 
animals.     They  form  tangled  slender  mycelia  with  many  spores  of 
varying  size.     They  occasion  "  ringworm,"  barber's  itch,  pityriasis, 
and  tinea.     Further  description  of  the  organisms  will  be  found  in  the 
section  upon  Ringworm. 


Fig. 


-Mucor  mucedo.     Single-celled  mycelium  with  three  hyphae  and  one 
developed  sporangium.     (After  Kny,  from  Tavel.) 


3.  Mucor.— -The  mucors,  or  " black  molds,"  belong  to  the  phyco- 
mycetes.  They  form  a  thick,  tangled  mycelium,  in  and  above  which 
the  rounded  black  sporangia  can  be  seen  with  the  naked  eye.  The 
mycelium  becomes  divided  at  the  time  of  reproduction.  Multiplica- 
tion takes  place  asexually  through  conidia-spores  which  develop 
within  sporangia,  and  sexually  by  the  conjugation  of  specialized 
terminal  septate  branches  of  the  mycelium,  which  conjugate  with 
similar  cells,  belonging  to  other  colonies,  to  form  zygospores. 

The  sporangia  form  upon  the  ends  of  aerial  hypha  and  consist  of  a 
smooth  spherical  capsule  within  which  the  spores  develop,  to  become 
liberated  only  when  the  membrane  ruptures.  The  colonies,  each  of 
which  is  unisexual,  may  be  described  as  +  and  — .  Colonies  of  the 
-f  type  will  not  conjugate;  colonies  of  the  —  type  will  not  conjugate, 
but  when  terminal  filaments  of  +  and  -  come  together,  conjuga- 
tion occurs  and  zygospore  formation  takes  place. 


42  Structure  and  Classification  of  Micro-organisms 

Mucors  are  not  infrequent  organisms  of  the  atmosphere  and 
occasionally  appear  as  contaminations  upon  solid  culture-media. 
About  130  species  are  known.  Of  these,  Mucor  corymbifer,  Mucor 
rhizopodiformis,  Mucor  ramosus,  Mucor  pusillus,  Mucor  septatus, 
and  Mucor  conoides  are  said  by  Plaut*  to  be  pathogenic  when 
introduced  into  laboratory  animals.  Mucor  ccgymbifer  has  been 
known  to  produce  inflammation  of  the  external 'auditory  meatus  in 
man.f  General  mucor  mycosis  in  man  has  also  been  observed  by 
Paltauf  t  to  result  from  the  presence  of  the  same  organism. 

4.  Aspergillus  and  Eurotium. — The  organisms  of  this  genus  are 
included  among  the  Ascomycetes.  They  are  common  organisms  of 


Fig.  12. — Mucor  mucedo.  Different  stages  in  the  formation  and  germination 
of  the  zygospore:  i,  Two  conjugating  branches  in  contact;  2,  septation  of  the 
conjugating  cells  (a)  from  the  suspensors  (6);  3,  more  advanced  stage  in  the 
development  of  the  conjugating  cells  (a);  4,  ripe  zygospore^  (ft)  between  the 
suspensors  (a);  5,  germinating  zygospore  with  a  germ-tube  bearing  a  sporangium. 
(After  Brefeld.) 

the  air  and  frequent  contaminations  of  solid  culture-media. 
To  secure  them  an  agar-agar  plate  can  be  exposed  to  the  atmosphere 
of  the  laboratory  for  a  short  time,  then  covered  and  stood  aside  for  a 
day  or  two,  when  tangled  mycelial  growths  with  rapidly  spreading 
hyphae  will  usually  be  discovered.  The  recognition  is  easily  made 
when  the  sporangia  appear.  These  are  well  shown  in  the  accom- 
panying illustration.  The  mycelium  is  divided  into  many  cells. 
Reproduction  is  asexual  and  takes  place  through  conidia  spores. 
The  fruit  hyphae,  which  are  aerial,  terminate  in  rounded  extremities 
which  are  known  as  columella,  from  which  many  radiating  sterig- 
mata  arise,  each  terminating  in  a  series  of  rounded  spores.  A  sexual 

*  Kolle  and  Wassermann,  "Die  Pathogenen  Mikroorganismen,"  1903,  i,  552. 
|  Hiickel-Losch  in  Fliigge,  "Die  Mikroorganismen."  %  Ibid. 


The  Molds 


form  of  reproduction  also  takes  place  through  the  production  of 
ascospores.  Many  species  are  known,  only  a  few  of  which  are 
pathogenic. 

Aspergillus  malignum  has  been  found  by  von  Lindt  in  the  auditory 
meatus  of  man. 

Aspergillus  nidulans  occasionally  infects  cattle.  It  is  pathogenic 
for  laboratory  animals,  usually  causing  death  in  sixty  hours.  The 
kidneys  are  found  enlarged  to  twice  thdf  normal  size,  and  show  small 
whitish  dots  and  stripes  of  cell  infiltration  containing  the  fungi. 


Fig.  13. — Aspergillus  glaucus:  A,  A  portion  of  the  mycelium  m,  with  a  con- 
idiaphore  c,  and  a  young  perithrecium  F,  magnified  190  diameters;  B  and  B', 
conidiaphore  with  conidia;  B,  individual  sterigma  greatly  magnified;  C,  early 
stage  of  the  development  of  the  fructifying  organ;  D,  young  perithrecium  in 
longitudinal  section;  w,  the  future  wall  of  the  contents;  as,  the  screw,  magnified 
250  diameters;  E,  an  ascus  with  spores  from  a  perithrecium,  magnified  600 
diameters.  (duBary.) 

The  heart  muscle,  diaphragm,  and  spleen  may  also  be  involved. 
The  liver  usually  escapes.  It  takes  a  large  number  of  spores  to 
infect. 

Aspergillus  fumigatus. — This  is  a  widespread  and  not  infrequently 
pathogenic  form.  Its  most  common  lesion  is  a  pneumomycosis,  in 
which  the  lung  is  riddled  with  small  inflammatory  necrotic  and 
cavernous  areas  containing  the  molds.  The  same  condition  has 
occasionally  been  observed  in  human  beings,  Sticker  having  collected 
39  cases.* 

Leber  and  others  have  observed  keratitis  following  corneal  infec- 
tion by  this  organism. 

Aspergillus  flavus  is  also  pathogenic. 

*  Nothnagel's  Spezielle  Path.  u.  Therap.,  xrv,  1900. 


44          Structure  and  Classification  of  Micro-organisms 

Aspergillus  subfuscus  is  also  pathogenic  and  highly  virulent. 

Aspergillus  niger. — Pathogenic  and  found  at  times  in  inflammation 
of  the  external  auditory  meatus. 

5.  Penicillium. — These  are  common  green  molds,  widely  dis- 
seminated throughout  the  atmosphere  and  frequent  sources  of 
contamination  of  the  culture-media  in  the  laboratory.  Moist  bread 
exposed  to  the  atmosphere  soon  becomes  covered  with  them.  They 
are  included  in  the  group  of  fungi  imperfecti,  and  are  characterized  by 
a  luxuriant  tangled  septate  mycelium,  with  aerial  fruit  hyphae, 
ending  in  conidiophores,  each  of  which  divides  into  two  or  three 
sterigmata,  the  tip  of  which  forms  a  chain  of  rounded  spores.  The 
whole  germinal  organ  thus  comes  to  resemble  a  whisk-broom  or,  as 


14. — Penicillium.     (Eyre.) 


Hiss  describes  it,  a  skeleton  hand,  in  which  the  conidiophore  cor- 
responds to  the  wrist;  the  sterigmata,  to  the  metacarpal  bones;  the 
chains  of  spores,  to  the  phalanges. 

None  of  the  penicillia  is  known  to  be  pathogenic  either  for  man  or 
animals. 

Penicillium  crustaceum  (glaucum)  is  the  most  common  source  of 
contamination  of  the  laboratory  media. 

Penicillium  minimum,  which  may  be  identical  with  the  preceding, 
was  once  found  in  the  human  ear  by  Sievenmann. 

THE  PROTOZOA 

The  protozoa  are  unicellular  animal  organisms  as  differentiated 
from  the  metazoa  which  are  multicellular  animal  organisms.  The 
restriction,  implied  by  the  term  unicellular  is,  however,  too  narrow, 
for  there  are  colonial  protozoa  that  consist  of  many  cells,  yet  share 
other  protozoan  characters. 

For  the  purposes  of  this  work,  however,  all  protozoa  are  to  be  re- 
garded as  unicellular  and  the  individuals  independent  of  one  another. 

Classification. — Many  schemes  have  been  devised  for  systematic- 
ally arranging  the  protozoa,  that  which  follows  being  an  abbrevia- 
tion of  the  standard  classification,  made  to  correspond  with  the 
requirements  of  this  work  that  deals  only  with  the  pathogenic  forms. 


The  Protozoa  45 

CLASSIFICATION  OF  THE  PATHOGENIC  PROTOZOA 

Phylum    PROTOZOA    (irpurros    first,    £uov     animal).      Unicellular     animal 

organisms. 

Class  Rhizopoda  (pit; a  root,  TrcoSos  foot).  Having  soft  plasmic  bodies 
with  or  without  external  protecting  shells.  The  contour  subject 
to  change  through  the  formation  of  extensions  known  as  pseudopods. 
These  may  be  blunt,  rounded,  or  lobose,  filamentous,  or  anastomosing. 
The  nutrition  is  holozoic  or  holophytic. 
Order  GYMNAMCEBA  (JV/JLVOS  naked).  Rhizopoda  without  external 

shells  or  coverings. 
Genus  Amoeba  (a/jiolpa  to  change). 
Genus  Entamceba. 
Genus  Chlamydophrys. 
Genus  Leydenia. 

Class  Mastigophora  (juao-Tryos  wmps,  <£dpos  to  bear).  Organisms  of 
well-defined  form,  naked  or  surrounded  by  a  well-defined  membrane. 
Nutrition  is  holozoic,  holophytic,  parasitic,  or  saprophytic.  Mouth, 
contractile  vesicle,  and  nucleus  usually  present. 

Order  FLAGELLATA   (Latin,  Jlagellare,  to  beat).     Small  organisms  with 

a  well-defined  mononucleate  body,  at  the  anterior  end  or  both  ends 

of  which  are  one  or  more  flagella.     Actively  motile.     May  become 

encysted.    Nutrition  is  holozoic,  holophytic,  parasitic,  or  saprophytic. 

Family  Ccrcomonidce.     Body  pyriform    with  several  anterior  flagella 

and  an  undulating  membrane. 
Genus  Cercomonas. 
Genus  Trichomonas. 
Genus  Monas. 
Genus  Plagiomonas. 

Family  LambliadcB.     Body  pyriform,  very  much  attenuated  behind. 
Ventral  surface  shows  a  reniform  depression,  about  the  posterior 
part  of  which  there  are  six  flagella.     There  are  also  two  flagella 
at  the  posterior  extremity. 
Genus  Lamblia  (Megastomum). 

Family  Trypanosomidce.  Body  delicately  fusiform.  Contains  a 
nucleus,  a  blepr  aroplast  or  centrosome,  and  an  undulating  mem- 
brane. A  single  wavy  flagellum  arises  in  the  posterior  part 
of  the  body  close  to  the  centrosome,  passes  along  the  edge  of  the 
undulating  membrane  to  the  anterior  extremity,  where  it  continues 
free  for  some  distance.  Nutrition  parasitic.  Reproduces  by 
division. 

Genus  Trypanosoma. 
Genus  Leishmania. 
Genus  Babesia. 

Family    Spirochcetidce.     Organisms    very    long    and    spirally    twisted. 
Nucleus    indistinct.     Multiplication    probably    by    longitudinal 
division  only.     Nutrition  is  parasitic  or  saprophytic. 
Genus  Spirochaeta.     Body  flattened,  with  a  very  narrow  undulating 

membrane. 
Genus  Treponema.     Body  not  flattened.     No  undulating  membrane. 

Extremities  sharp  pointed  and  terminating  in  short  flagella. 
Class  Sporozoa  (o-Tropos  a  spore,  $uov  an  animal).     Organisms  unprovided 
with  cilia  or  flagella  in  the  adult  stage.     Always  endoparasites  in  the 
cells,  tissues,  or  cavities  of  other  animals.     Nutrition  is  parasitic  and 
osmotic.     Reproduction   always   by  spore-formation,  the  sporozoites 
either  being  produced  by  the  parent  or  indirectly  from  spores,  into 
which  the  parent  divides. 
Subclass    Telosporidia.     Spore-formation    ends    the  individual    life,  the 

entire  organism  being  transformed  to  spores. 

Order  GREGARINIDA.  Possess  distinct  membrane  with  myonemes 
during  adult  life;  locomotion  mainly  by  contraction.  Young  stages 
alone  (cephalonts)  are  intracellular  parasites,  the  adults  (sporonts) 
being  found  in  the  digestive  tract  or  the  body  cavities.  Sporulation 
takes  place  after  or  without  conjugation,  but  within  a  cyst  that  is 
never  formed,  while  the  parasite  is  intracellular. 


46          Structure  and  Classification  of  Micro-organisms 

Order  COCCIDIIDA.     Spherical  or  ovoid  in  form,  without  a  free  and 
motile   adult   stage.     Never   ameboid.     Sporulation   takes   place 
within  cysts  formed  while  the  organism  is  an  intracellular  parasite. 
Genus  Coccidium. 
Genus  Eimeria. 

Order  H^MOSPORIDIIDA.     Sporozoa  of  small  size  living  in  the  blood- 
corpuscles  or  plasma  of  vertebrates.     The  adult  form  is  mobile 
and  in  some  cases  provided  with  myonemes.     Reproduction  by 
endogenous  or  asexual  sporulation,  while  in  the  host  or  by  ex- 
ogenous sporulation  after  conjugation. 
Genus  Plasmodium. 
Subclass  Neosporidia.     Organisms  that  form  sporocysts  throughout  life, 

the  entire  cell  not  being  used  up  in  the  formation  of  the  spores. 
Order  SARCOSPORIDIA.     The  initial  stage  of  the  life  history  is  passed 
in  the  muscle  cells  of  vertebrates.     Form  is  elongate,   tubular, 
oval,  or  even  spherical.     Cysts  have  a  double  membrane,  in  which 
reniform  or  falciform  sporozoites  are  formed. 
Genus  Sarcocystis. 
Genus  Miescheria. 
Genus  Balbiania. 
Subclass  Haplosporidia.     Spores  provided  with  large  round  nuclei.     No 

polar  capsules. 
Genus  Rhinosporidium. 

Class  Infusoria  (Latin,  infusus,  to  pour  into.  The  organisms  were  given 
this  name  because  they  were  first  found  in  infusions  exposed  to  the 
air).  Protozoa  in  which  the  motor  apparatus  is  in  the  form  of  cilia, 
either  simple  or  united  into  membranes,  membranelles,  or  cirri.  The 
cilia  may  be  permanent  or  limited  to  the  embryonic  stages.  There 
are  two  kinds  of  nuclei,  macronucleus  and  micronucleus.  Reproduc- 
tion is  effected  by  simple  transverse  division  or  by  budding.  Nutrition 
is  holozoic  or  parasitic. 
Subclass  Ciliata.  Mouth  and  anus  usually  present.  The  contractile 

vacuole  often  connected  with  a  complicated  system  of  canals. 
Order  HOLOTRICHIDA.     The  cilia  are  similar  and  distributed  all  over 
the  body,  with  a  tendency  to  lengthen  at  the  mouth.     Trichocysts 
are  always  present,  either  over  the  whole  body  or  in  special  regions. 
Genus  Colpoda. 
Genus  Chilodon. 

Order    HETEROTRICHIDA.     Organisms    possessing  a  uniform  covering 
of  cilia  over  the  entire  body,  and  an  adoral  zone  consisting  of  short 
cilia  fused  together  into  membranelles. 
Suborder  Polytrichina.     Uniform  covering  of  cilia. 

Family  Bursar  idee.  The  body  is  usually  short  and  pocketlike, 
but  may  be  elongated.  The  chief  characteristic  is  the 
peristome,  which  is  not  a  furrow,  but  a  broad  triangular  area 
deeply  insunk,  and  ending  in  a  point  at  the  mouth.  The 
adoral  zone  is  usually  confined  to  the  left  peristome  edge  or 
it  may  cross  over  to  the  right  anterior  edge. 
Genus  Balantidium. 

Structure. — From  the  table  it  will  at  once  be  evident  that  the 
protozoa  form  an  extremely  varied  group,  and  that  no  kind  of 
descriptive  treatment  can  be  looked  upon  as  adequate  that  does  not 
consider  individuals. 

Cytoplasm. — In  some  of  the  smaller  protozoa,  and  in  certain  stages 
of  others,  the  cytoplasm  appears  almost  hyaline  and  structureless. 
In  most  cases,  however,  it  appears  granular,  and  in  the  larger  organ- 
isms, such  as  ameba,  it  presents  the  appearance  which  some  described 
as  granular,  others,  as  frothy.  The  accepted  theory  of  structure 
teaches  that  the  protoplasm  is  honeycombed  or  frothy,  and  that  it  is 


The  Protozoa 


47 


filled  with  endless  chambers  in  which  its  enzymes  and  other  active 
substances,  etc.,  are  stored  up  and  its  functions  carried  on. 

In  addition  to  these  chambers,  which  are  minute  and  of  uniform 
size,  there  are  larger  spaces  called  vacuoles,  some  of  which  are  the 
result  of  temporary  conditions — accumulations  of  digested  but  not 
yet  assimilated  food,  etc.;  but  others,  seen  in  ameba  and  in  the 
ciliata,  are  large,  permanent,  and  characterized  by  rhythmical 
contractions  through  which  they  disappear  from  one  part  of  the 
body  substance  to  appear  in  another.  These  are  known  as  "  con- 
tractile vacuoles,"  and  are  supposed  to  subserve  the  useful  purpose  of 
assisting  in  maintaining  cytoplasmic  currents  and  so  distributing  the 
nourishing  juices. 

The  cytoplasm  also  contains  remnants  of  undigested  or  indigest- 
ible foods  which  constitute  the  paraplasm  or  deuteroplasm.  In  a 


D 


Fig.  15. — Internal  parasites:  A,  Amoeba  coli,  Losch;  B,  Monocystis  agilis, 
Leuck.,  a  gregarine;  C,  Megastoma  entericum,  Grassi,  a  flagellate;  D,  Balantidium 
coli,  Ehr.,  a  ciliate. 

few  cases  granules  of  chlorophyl  are  also  to  be  found  in  organisms 
otherwise  resembling  animals  too  closely  to  be  confused  with  plants. 

The  cytoplasm  may  be  soft  and  uniform  in  quality,  or  there  may 
be  a  surface  differentiation  into  ectosarc,  or  body  covering,  and 
endosarc,  body  substance.  In  the  rhizopoda  there  is  little  difference 
between  the  two,  though  certain  fresh-water  ameba  cover  themselves 
with  minute  grains  of  mineral  substance,  but  in  most  of  the  masti- 
gophora  and  infusoria  corticata  the  ectosarc  is  characterized  by  a 
peculiar  rigidity  that  gives  the  animal  a  definite  and  permanent 
form.  From  the  surface  covering  or  ectosarc  coarse  threads  or  fine 
hair-like  appendages— flagella  and  cilia — often  project.  In  many 
of  the  infusoria  the  ectosarc  contains  trichocysts  from  which  nettling 
or  stinging  threads  are  thrown  out  when  the  organisms  are  irritated. 

The  body  substance  may  show  no  morphologic  differentiation  in 
rhizopoda,  but  in  the  corticata  there  may  not  only  be  a  permanent 


48  Structure  and  Classification  of  Micro-organisms 

form,  but  there  may  be  adaptations,  such  as  an  oral  aperture,  some- 
times infundibular  in  shape  and  communicating  with  the  soft 
endosarc  through  a  blind  tube.  An  anal  aperture  may  also  be 
present. 

In  the  higher  infusoria  the  ectosarc  may  also  be  continued  pos- 
teriorly to  form  a  stalk,  by  which  the  organism  attaches  itself 
(Vorticella).  Such  stalks  are  contractile. 

Nucleus. — In  certain  protozoa  of  very  simple  and  indefinite 
structure — spirochaeta  and  treponema — no  distinct  well-contoured 
nucleus  can  be  observed. 

In  the  rhizopoda  the  nucleus  is  a  distinct  organ  surrounded  by  a 
nuclear  membrane  and  containing  the  usual  chromatin  and  linin. 

The  greater  number  of  mastigophora  possess  two  distinct  bodies, 
either  a  nucleus  and  a  centrosome  or  a  major  and  minor  nucleus. 
This  is  well  shown  in  trypanosoma. 

The  infusoria  vary  greatly  in  the  character  of  the  nuclei.  As  a 
rule,  there  are  two  indefinite  nuclei,  the  macronucleus  and  the 
micronucleus.  Both  seem  to  be  essential  organs,  and  in  the  phe- 
nomena supervening  upon  conjugation  both  participate.  The 
nuclei  of  the  protozoa  are,  therefore,  extremely  diversified,  and 
vary  from  the  most  simple  collections  of  granules  of  nuclear  sub- 
stance to  large  well-formed  fantastically  shaped  composite  organs. 

Movement. — Some  kind  of  movement  is  to  be  observed  at  some 
period  in  the  life  of  almost  every  protozoan. 

In  rhizopoda  with  the  soft  ectosarc  the  movement  consists  of 
flowing  currents  by  which  lobose  projections  of  the  body  substance 
appear  now  here,  now  there,  in  the  form  of  pseudopodia,  or  else  a 
continuous  flowing,  by  which  the  upper  surface  continually  coming 
forward  in  a  thin  layer  coincides  with  the  progress  of  the  animal, 
which  continually  rolls  over  and  over  as  it  were. 

In  mastigophora  the  movement  of  the  more  rigid  bodies  is  effected 
through  the  presence  of  longer  or  shorter,  flexile  or  rigid,  coarse 
threads  or  "  whips."  These  usually  project  anteriorly — trypano- 
soma— and  by  means  of  a  spiral  movement  draw  the  cell  along  with 
a  propeller-like  action;  symmetrically  arranged  flagella  may  operate 
more  like  oars. 

The  sporozoa  usually  manifest  very  little  movement,  yet  their 
sporozoiites  are  motile,  and  the  spermatozoites  are  also  motile  and 
commonly  flagellated. 

The  infusoria  are  actively  motile  through  abundant  fine  hair-like 
formations  known  as  cilia.  These,  multitudinous  as  they  are, 
vibrate  synchronously  with  an  oar-like  movement,  propelling  the 
organisms  forward  or  backward  or  making  them  revolve  with  great 
rapidity.  Independent  cilia  not  infrequently  encircle  the  oral 
aperture,  causing  a  vortex,  in  which  the  minute  structures  upon 
which  the  creatures  feed  are  caught  and  carried  into  the  body. 

Size. — The  protozoa  show  very  great  variation  in  size.     Some  of 


The  Protozoa  49 

the  sporozoa  for-m  minute  parasites  of  the  red  blood-corpuscles  or 
other  cells  of  the  vertebrates.  The  treponema  is  so  small  that  it 
can  slowly  find  its  way  through  the  pores  of  a  Berkefeld  filter. 

On  the  other  hand,  the  sarcoporidium  is  so  large  that  one  of  its 
cysts,  composed  of  a  single  organism,  can  be  seen  with  the  naked 
eye.  Certain  protozoa  that  play  no  part  in  morbid  processes — 
myxosporidia — and  so  do  not  come  within  the  scope  of  this  work, 
may  be  several  centimeters  in  diameter. 

Reproduction. — The  reproduction  of  the  protozoa  takes  place  both 
asexually  and  sexually.  It  may  be  that  there  are  no  strictly  asexual 
protozoa,  nearly  all  forms  having  been  shown  upon  intimate  ac- 
quaintance to  be  subject  to  occasional  conjugation.  Conjugation 
may  result  in  the  loss  of  individual  identity  or  the  conjugated 
individuals  may  again  separate. 

Whether  the  reproduction  takes  place  asexually  without  con- 
jugation or  sexually  after  conjugation,  it  always  occurs  by  division, 
which  may  be  simple  and  binary  or  complex  and  multiple. 

Wherever  a  distinct  nucleus  can  be  found,  the  multiplication  of  the 
protozoa  is  preceded  by  some  kind  of  mitotic  change.  The  more 
complex  the  structure  of  the  nucleus,  the  more  complicated  and 
perfect  the  mitosis. 

The  elongate  protozoa  divide  lengthwise,  which  is  sometimes 
contrary  to  expectation,  as  in  the  cases  of  treponema  and  spirochaeta. 

The  multitudinous  sporozo'ites  into  which  the  zygotes  of  the 
sporozoa  divide  are  commonly  the  result  of  anterior  division  into 
intermediate  bodies  known  as  oocysts,  ookinetes,  sporocysts,  etc. 
The  nuclear  substance  is  first  divided  so  as  to  be  uniformly  dis- 
tributed among  these,  then  further  divided  so  that  some  of  it  reaches 
each  sporozoite. 

In  the  process  of  sporulation  the  entire  parent  may  be  used  up, 
as  in  the  coccidium  and  plasmodium  or  the  parent  may  continue  to 
live  and  later  form  additional  sporozo'ites,  as  in  sarcocystis. 

Encystment. — Nearly  all  of  the  protozoa  are  capable  at  times  of 
encysting  themselves,  i.e.,  surrounding  themselves  with  dense 
capsules  by  which  life  may  be  preserved  for  some  time  amid  such 
unfavorable  surroundings  as  excessive  cold,  excessive  dryness,  and 
absence  of  food.  Sometimes  the  encysted  stage  is  the  spore  stage 
(coccidium),  sometimes  it  is  the  adult  stage  (ameba).  Under  these 
circumstances  we  find  an  analogy  with  the  sporulation  of  the 
bacteria  which  is  not  for  purposes  of  multiplication,  but  for  self- 
preservation.  The  encysted  protozoa  are  less  hardy,  however,  than 
the  bacterial  and  other  plant  spores,  and  succumb  to  comparatively 
slight  elevations  of  temperature. 


CHAPTER  II 
BIOLOGY  OF  MICRO-ORGANISMS 

THE  distribution  of  micro-organisms  is  well-nigh  universal.  They 
and  their  spores  pervade  the  atmosphere  we  breathe,  the  water  we 
drink,  the  food  we  eat,  and  luxuriate  in  the  soil  beneath  our  feet. 

They  are  not,  however,  ubiquitous,  but  correspond  in  distribution 
with  that  of  the  matter  upon  which  they  live  and  the  conditions 
they  can  endure.  Tyndall*  found  the  atmosphere  of  high  Alpine 
altitudes  free  from  them,  and  likewise  that  the  glacier  ice  contained 
none;  but  wherever  man,  animals,  or  plants  live,  die,  and  decom- 
pose, they  are  sure  to  be. 

Their  presence  in  the  air  generally  depends  upon  their  previous 
existence  in  the  soil,  its  pulverization,  and  distribution  by  currents 
of  the  atmosphere.  Koch  has  shown  that  the  upper  stratum  of  the 
soil  is  exceedingly  rich  in  bacteria,  but  that  their  numbers  decrease 
as  the  soil  is  penetrated,  until  below  a  depth  of  one  meter  there  are 
very  few.  Remembering  that  micro-organisms  live  chiefly  upon 
organic  matter,  this  is  readily  understandable,  as  most  of  the  organic 
matter  is  upon  the  surface  of  the  soil.  Where,  as  in  the  case  of 
porous  soil  or  the  presence  of  cesspools  and  dung-heaps,  the  de- 
composing materials  are  allowed  to  penetrate  to  a  considerable 
depth,  micro-organisms  may  occur  much  farther  below  the  surface; 
yet  they  are  rarely  found  at  any  great  depth,  because  the  majority 
of  them  require  free  oxygen  for  successful  existence. 

The  water  of  stagnant  pools  always  teems  with  micro-organisms; 
that  of  deep  wells  rarely  contains  many  unless  it  is  polluted  from  the 
surface  of  the  earth. 

It  has  been  suggested  by  Soyka  that  currents  of  air  passing  over 
the  surface  of  liquids  might  take  up  organisms,  but,  although  he 
seemed  to  show  it  experimentally,  it  is  not  generally  believed. 
Where  bacteria  are  growing  in  colonies  they  seem  to  remain  un- 
disturbed by  currents  of  air  unless  the  surface  of  the  colony  becomes 
roughened  or  broken. 

Most  of  the  organisms  carried  about  by  the  air  are  what  are  called 
saprophytes,  and  are  harmless. 

Oxygen. — As  all  micro-organisms  must  have  oxygen  in  order  to 
live,  the  greater  number  of  them  grow  best  when  freely  exposed  to 
the  air.  Some  will  not  grow  at  all  where  uncombined  oxygen  is 
present,  but  secure  all  they  need  by  severing  it  from  its  chemic 
combinations.  These  peculiarities  divide  bacteria  into  the 
*  "Floating  Matter  in  the  Air." 


Conditions  Prejudicial  to  Growth  of  Bacteria  51 

Aerobes,  which  grow  in  the  presence  of  uncombined  oxygen,  and 

Anaerobes,  which  do  not  grow  in  the  presence  of  uncombined 
oxygen. 

As,  however,  some  of  the  aerobic  forms  grow  almost  as  well  with- 
out free  oxygen  as  with  it,  they  are  known  as  optional  (facultative) 
anaerobes, 

As  examples  of  strictly  aerobic  bacteria  Bacillus  subtilis,  Bacillus 
aerophilus,  Bacillus  tuberculosis,  and  Bacillus  diphtherias  may  be 
given.  These  will  not  grow  if  oxygen  is  denied  them.  The  cocci  of 
suppuration,  the  bacillus  of  typhoid  fever,  and  the  spirillum  of  cholera 
grow  almost  equally  well  with  or  without  free  oxygen,  and  hence 
belong  to  the  optional  anaerobes.  The  bacilli  of  tetanus  and  of 
malignant  edema  and  the  non-pathogenic  Bacillus  butyricus, 
Bacillus  muscoides,  and  Bacillus  polypiformis,  will  not  develop  at 
all  where  any  free  oxygen  is  present,  and  hence  are  strictly  anaerobic. 

The  higher  bacteria,  o'idia,  molds  and  protozoa,  are  for  the  most 
part  aerobesand  optional  anaerobes.  Treponema  pallidum  seems  to 
be  a  strictly  anaerobic  protozoan. 

Food. — The  bacteria  grow  best  where  diffusible  albumins  are 
present,  the  ammonium  salts  being  less  fitted  to  support  them  than 
their  organic  compounds.  Proskauer  and  Beck*  have  succeeded 
in  growing  the  tubercle  bacillus  in  a  mixture  containing  ammonium 
carbonate  0.35  per  cent.,  potassium  phosphate  0.15  per  cent.,  mag- 
nesium sulphate  0.25  per  cent.,  and  glycerin  1.5  per  cent.  Some  of 
the  water  microbes  can  live  in  distilled  water  to  which  the  smallest 
amount  of  organic  matter  has  been  added;  others  require  so  con- 
centrated a  medium  that  only  blood-serum  can  be  used  for  their 
cultivation.  The  statement  that  certain  forms  of  bacteria  can 
nourish  in  clean  distilled  water  seems  to  be  untrue,  as  in  this 
medium  the  organisms  soon  die  and  disintegrate.  If,  however,  in 
making  the  transfer,  a  drop  of  culture  material  is  carried  into  the 
water  with  the  bacteria,  the  distilled  water  ceases  to  be  such,  and 
becomes  a  diluted  bouillon  fitted  to  support  bacterial  life  for  a  time. 
Sometimes  a  species  with  a  preference  for  a  particular  culture  medium 
can  gradually  be  accustomed  to  another,  though  immediate  trans- 
plantation causes  the  death  of  the  organism.  Sometimes  the  addi- 
tion of  such  substances  as  glucose  and  glycerin  has  a  peculiarly 
favorable  influence,  the  latter,  for  example,  enabling  the  tubercle 
bacillus  to  grow  upon  agar-agar. 

The  yeasts  grow  best  upon  media  containing  sugars,  but  can  also 
be  cultivated  upon  media  containing  diffusible  protein  and  non- 
fermentable  carbohydrates  and  glycerin. 

The  molds  flourish  upon  almost  all  kinds  of  organic  matter,  but 
perhaps  attain  their  most  rapid  development  upon  media  containing 
fermentable  carbohydrates. 

*  "Zeitschrift  fur  Hygiene,''  etc.,  Aug.  10,  1894,  vol.  xvin,  No.  i. 


52  Biology  of  Micro-organisms 

The  saprophytic  and  parasitic  protozoa  live  by  osmosis  and  absorb 
through  the  ectosarc  such  substances  as  are  capable  of  assimilation 
and  nutrition.  These  forms  are  cultivable  only  upon  media  con- 
taining the  same  or  approximately  the  same  proteins  as  those  to 
which  they  have  been  accustomed.  Thus,  to  cultivate  trypanosoma, 
blood-serum  must  be  added  to  the  media. 

The  larger  protozoa  live  upon  smaller  animal  and  vegetable  organ- 
isms, which  they  ingest  entire.  Such  can  only  be  artificially  culti- 
vated provided  the  attempt  be  made  under  conditions  of  symbiosis 
with  some  other  and  smaller  organism  that  may  constitute  the 
food. 

Moisture. — A  certain  amount  of  water  is  indispensable  to  the 
growth  of  bacteria.  The  amount  can  be  exceedingly  small,  however, 
Bacillus  prodigiosus  being  able  to  develop  successfully  upon  crackers 
and  dried  bread.  Artificial  culture-media  should  not  be  too  con- 
centrated; at  least  80  per  cent,  of  water  should  be  present. 

The  molds  and  o'idia  grow  well  upon  bread  that  contains  very 
little  moisture.  Protozoa  usually  require  fluid  media.  Pond-water 
protozoa  can  only  grow  in  wrater,  not  in  concentrated  culture-media. 

Reaction. — Should  the  pabulum  supplied  contain  an  excess  of 
either  alkali  or  acid,  the  growth  of  the  micro-organisms  is  inhibited. 
Most  true  bacteria  grow  best  in  a  neutral  or  feebly  alkaline  medium. 
There  are  exceptions  to  this  rule,  however,  for  Bacillus  butyricus  and 
Sarcina  ventriculi  can  grow  well  in  strong  acids,  and  Micrococcus 
urea  can  tolerate  excessive  alkalinity.  Acid  media  are  excellent 
for  the  cultivation  of  molds.  Neutral  or  feebly  alkaline  media  serve 
best  for  the  cultivable  protozoa. 

Light. — Most  organisms  are  not  influenced  by  the  presence  or 
absence  of  ordinary  diffused  daylight.  The  direct  rays  of  the  sun, 
and  to  a  less  degree  the  rays  of  the  electric  arc-light,  retard  and  in 
numerous  instances  kill  bacteria.  In  a  careful  study  of  this 
subject  Weinzirl*  found  that  when  bacteria  were  placed  upon  glass 
or  paper,  and  exposed  to  the  direct  rays  of  the  sun,  without  any 
covering,  most  non-spore-bearing  bacteria,  including  Bacillus  tubercu- 
losis, B.  diphtherias,  B.  typhosus,  S.  choleras  asiaticae,  B.  coli,  B. 
prodigiosus,  and  others  are  killed  in  from  two  to  ten  minutes. 
Certain  colors  are  distinctly  inhibitory  to  the  growth,  blue  being 
especially  prejudicial. 

Treskinskajaf  found  that  sunlight  had  a  marked  destructive  effect 
upon  the  tubercle  bacillus,  and  varied  according  to  altitude.  By 
direct  sunlight  at  the  sea-level  they  were  destroyed  in  five  hours:  at 
an  altitude  of  1560  meters,  in  three  hours.  In  winter  the  time  of 
destruction  was  about  two  hours  longer  than  in  summer.  In  diffused 
daylight  the  time  required  for  destruction  was  about  twice  as  long 

*"Centralbl.  f.  Bakt.  u.  Parasitenk.  Ref.,"  XLVII,  Nos.  22-24,  P-  681. 
t"Jour~  Infectious  Diseases,"   1907,  vol.  iv,  Supplement,  No.  3,  p.  128. 


Conditions  Prejudicial  to  Growth  of  Bacteria          53 

as  in  direct  sunlight.  His  experiments  were  performed  with  pure 
cultures  dried  in  a  thin  layer  upon  glass. 

Certain  chromogenic  bacteria  produce  colors  only  when  exposed 
to  the  ordinary  light  of  the  room.  Bacillus  mycoides  roseus  produces 
its  red  pigment  only  in  the  dark.  The  virulence  of  many  pathogenic 
bacteria  is  gradually  attenuated  if  they  are  kept  in  the  light. 

Molds  and  yeasts  grow  best  in  the  dark,  so  that  in  general  it  can 
be  said  that  the  vegetable  micro-organisms,  belonging  to  the  fungi 
and  having  no  chlorophyl,  need  no  light  and  are  injured  rather 
than  benefited  by  it. 

The  pathogenic  protozoa  have  not  been  particularly  studied 
with  reference  to  light.  Non-pathogenic  water  protozoa  love  the 
light  and  die  in  the  dark. 

Electricity,  X-rays,  etc. — Powerful  currents  of  electricity  passed 
through  cultures  have  been  found  to  kill  the  organisms  and  change 
the  reaction  of  the  culture-medium;  rapidly  reversed  currents  of  high 
intensity,  to  destroy  the  pathogenesis  of  the  bacteria  and  transform 
their  toxic  products  into  neutralizing  bodies  (antitoxin?).  Atten- 
tion has  been  called  to  this  subject  by  Smirnow,  d'Arsonval  and 
Charin,  Bolton  and  Pease,  Bonome  and  Viola,  and  others. 

An  interesting  contribution  upon  the  "Effect  of  Direct,  Alter- 
nating, Tesla  Currents  and  X-rays  on  Bacteria"  was  made  by  Zeit,* 
whose  conclusions  are  as  follows: 

1.  A  continuous  current  of  260  to  320  milliamperes  passed  through  bouillon 
cultures  kills  bacteria  of  low  thermal  death-points  in  ten  minutes  by  the  pro- 
duction of  heat   (g8.5°C).     The  antiseptics  produced  by  electrolysis  during 
this  time  are  not  sufficient  to  prevent  the  growth  of  even  non-spore-bearing 
bacteria.     The  effect  is  a  purely  physical  one. 

2.  A  continuous  current  of  48  milliamperes  passed  through  bouillon  cultures 
for  from  two  to  three  hours  does  not  kill  even  non-resistant  forms  of  bacteria. 
The  temperature  produced  by  such  a  current  does  not  rise  above  3 7°C.,  and  the 
electrolytic  products  are  antiseptic,  but  not  germicidal. 

3.  A  continuous  current  of  100  milliamperes  passed  through  bouillon  cultures 
for  seventy-five  minutes  kills  all  non-resistant  forms  of  bacteria  even  if  the 
temperature  is  artificially  kept  below  37°C.     The  effect  is  due  to  the  formation 
of  germicidal  electrolytic  products  in  the  culture.     Anthrax  spores  are  killed  in 
two  hours.     Subtilis  spores  were  still  alive  after  the  current  was  passed  for  three 
hours. 

4.  A  continuous  current  passed  through  bouillon  cultures  of  bacteria  produces 
a  strongly  acid  reaction  at  the  positive  pole,  due  to  the  liberation  of  chlorin 
which  combines  with  oxygen  to  form  hypochlorous  acid.     The  strongly  alkaline 
reaction  of  the  bouillon  culture  at  the  negative  pole  is  due  to  the  formation  of 
sodium  hydroxid  and  the  liberation  of  hydrogen  in  gas  bubbles.     With  a  current 
of  100  milliamperes  for  two  hours  it  required  8.82  milligrams  of  H2SO4  to  neutral- 
ize i  cc.  of  the  culture  fluid  at  the  negative  pole,  and  all  the  most   resistant 
forms  of  bacteria  were  destroyed  at  the  'positive  pole,  including  anthrax  and 
subtilis  spores.     At  the  negative  pole  anthrax  spores  were  killed  also,  but  subtilis 
spores  remained  alive  for  four  hours. 

5.  The  continuous  current  alone,  by  means  of  Du  Bois-Reymond's  method 
of  non-polarizing  electrodes,  and  exclusion  of  chemic  effects  by  ions  in  Kruger's 
sense,  is  neither  bactericidal  nor  antiseptic.     The  apparent  antiseptic  effect 
on  suspension  of  bacteria  is  due  to  electric  osmosis.     The  continuous  electric 
current  has  no  bactericidal  nor  antiseptic  properties,  but  can  destroy  bacteria 

*  "Jour.  Amer.  Med.  Assoc.,"  Nov.  30,  1901. 


54  Biology  of  Micro-organisms 

only  by  its  physical  effects  (heat)  or  chemic  effects  (the  production  of  bactericidal 
substances  by  electrolysis). 

6.  A  magnetic  field,  either  within  a  helix  of  wire  or  between  the  poles  of  a 
powerful  electromagnet,  has  no  antiseptic  or  bactericidal  effects  whatever. 

7.  Alternating  currents  of  a  3-inch  Ruhmkorff  coil  passed  through  bouillon 
cultures  for  ten  hours  favor  growth  and  pigment  production. 

8.  High-frequency,    high   potential   currents — Tesla   currents— have   neither 
antiseptic  nor  bactericidal  properties  when  passed  around  a  bacterial  suspension 
within  a  solenoid.     When  exposed  to  the  brush  discharges,  ozone  is  produced 
and  kills  the  bacteria. 

9.  Bouillon  and  hydrocele-fluid  cultures  in  test-tubes  of  non-resistant  forms 
of  bacteria  could  not  be  killed  by  Rontgen  rays  after  forty-eight  hours'  exposure 
at  a  distance  of  20  mm.  from  the  tube. 

10.  Suspensions  of  bacteria  in  agar  plates  and  exposed  for  four  hours  to  the 
rays,  according  to  Rieder's  plan,  were  not  killed. 

1 1.  Tubercular  sputum  exposed  to  the  Rontgen  rays  for  six  hours,  at  a  distance 
of  20  mm.  from  the  tube,  caused  acute  miliary  tuberculosis  of  all  the  guinea-pigs 
inoculated  with  it. 

12.  Rontgen  rays  have  no  direct  bactericidal  properties.     The  clinical  results 
must  be  explained  by  other  factors,  possibly  the  production  of  ozone,  hypochlor- 
ous  acid,  extensive  necrosis  of  the  deeper  layers  of  the  skin,  and  phagocytosis. 
The  action  of  the  z-rays  upon  bacteria  has  been  investigated  by  Bonome  and 
Gros,*  Pott,f  and  others.     When  the  cultures  are  exposed  to  their  action  for 
prolonged  periods,  their  vitality  and  virulence  seem  to  be  slightly  diminished. 
They  are  not  killed  by  the  #-rays. 

Movement. — Rest  seems  to  be  the  condition  best  adapted  for 
micro-organismal  development.  Slow-flowing  movements  do  not 
have  much  inhibitory  action,  but  violent  agitation,  as  by  shaking  a 
culture  in  a  machine,  may  hinder  or  prevent  it.  This  explains  why 
rapidly  flowing  streams,  whose  currents  are  interrupted  by  falls  and 
rapids,  should,  other  things  being  equal,  furnish  a  better  drinking- 
water  than  a  deep,  still-flowing  river. 

Galli-ValerioJ  has  shown,  however,  that  agitation  does  not  in- 
hibit the  growth  of  the  anthrax,  typhoid  or  colon  bacilli  or  the 
pneumococcus,  but  sometimes  facilitates  it. 

Association. — Symbiosis  is  the  vital  association  of  different  species 
of  micro-organisms  by  which  mutual  benefit  to  one  or  the  other  is 
brought  about.  Antibiosis  is  an  association  detrimental  to  one  of 
the  associated  organisms.  Bacterial  growth  is  greatly  modified  by 
the  association  of  different  species.  Coley  found  the  streptococcus 
more  active  when  combined  with  Bacillus  prodigiosus;  Pawlowski, 
that  mixed  cultures  of  Bacillus  anthracis  and  Bacillus  prodigiosus 
were  less  virulent  than  pure  cultures  of  anthrax;  Meunier,  §  that 
when  the  influenza  bacillus  of  Pfeiffer  is  inoculated  upon  blood  agar 
together  with  Staphylococcus  aureus  its  growth  is  favored  by  a 
change  which  the  staphylococci  bring  about  in  the  hemoglobin. 

A  similar  advantageous  association  has  been  pointed  out  by 
Sanarelli,  who  found  that  Bacillus  icteroides  grows  best  and  retains 

*"Giornal.  med.  del  Regis  Esercito,"  an  45,  u.  6. 
t  "Lancet,"  1897,  vol.  n,  No.  21. 

|  "Centralbl.  f.  Bakt.,"  etc.,  Sept.  23,  1904,  Orig.,  xxxvn,  p.  151. 
§  Societe  de  Biologic,  Seance  du  n  Juin,  1898,  "La  Semaine  medicale,"  June 
15,  1898. 


Conditions  Prejudicial  to  Growth  of  Bacteria  55 

its  vitality  longest  when  grown  in  company  with  certain  of  the 
molds. 

Rarely,  the  presence  of  one  species  of  micro-organism  entirely 
eradicates  another.  Hankin*  found  that  Micrococcus  ghadialli 
destroyed  the  typhoid  and  colon  bacilli,  and  suggested  the  use  of  this 
coccus  to  purify  waters  polluted  with  typhoid. 

An  interesting  experimental  study  of  the  bacterial  antagonisms 
with  special  reference  to  Bacillus  typhosus,  that  the  student 
should  read,  is  by  W.  D.  Frost,  and  appears  in  the  "  Journal  of 
Infectious  Diseases,"  1904,  i,  p.  599. 

Temperature. — According  to  Frankel,  bacteria  will  rarely  grow 
below  1 6°  and  above  4o°C.,  but  Fliigge  has  shown  that  Bacillus 
subtilis  will  grow  very  slowly  at  6°C.;  at  i2.5°C.  fission  does  not 
take  place  oftener  than  every  four  or  five  hours;  at  25°C.  fission 
occurs  every  three-quarters  of  an  hour,  and  at  3o°C.  about  every 
half-hour. 

The  temperature  at  which  micro-organisms  grow  best  is  known  as 
the  optimum,  the  lowest  temperature  at  which  they  continue  active 
as  the  minimum,  the  highest  that  can  be  endured  the  maximum. 

A  few  forms  of  bacteria  grow  at  very  high  temperatures  (6o°- 
7o°C.),  and  are  described  as  thermophilic.  They  are  found  in 
manure  piles  and  in  hot  springs.  Tsiklinskyf  has  described  two 
varieties  of  actinomyces  and  a  mold  that  he  cultivated  from  earth 
and  found  able  to  grow  well  at  48°  to  68°C.,  though  not  at  all  at  the 
temperature  of  the  room. 

Most  bacteria  are  killed  by  temperatures  above  60°  to  75°C.,  but 
their  spores  can  resist  boiling  water  for  some  minutes,  though  killed 
by  dry  heat  if  exposed  to  i5o°C.  for  an  hour  or  to  i75°C.  for  from 
five  to  ten  minutes. 

The  resistance  of  low  forms  of  life  to  low  temperatures  is  most 
astonishing.  Some  adult  bacteria  and  most  spores  seem  capable  of 
resisting  almost  any  degree  of  cold.  RavenelJ  exposed  anthrax 
spores  to  the  action  of  liquid  air  for  three  hours;  diphtheria  bacilli, 
for  thirty  minutes;  typhoid  bacilli,  for  sixty  minutes;  and  Bacillus 
prodigiosus,  for  sixty  minutes,  the  temperature  of  the  cultures  being 
reduced  to  about  — i4o°C.,  yet  in  no  case  was  the  vegetative  ca- 
pability of  all  of  the  bacteria  destroyed,  and  when  transferred  to  fresh 
culture  bouillon  they  grew  normally.  His  researches  corroborate 
those  of  Pictet  and  Yung  and  others. 

To  say  that  bacteria  are  not  injured  by  cold  is  a  mistake,  as 
Sedgwick  and  Winslow§  have  found  that  when  typhoid  bacilli  are 
frozen,  the  greater  number  of  them  are  destroyed,  and  that  subsequent 

*  "Brit.  Med.  Jour.,"  Aug.  14,  1897,  p.  418. 

"Russ.  Archiv  f.  Path.,"  etc.,  June,  1898,  Bd.  v. 
j  "The  Medical  News,"  June  10,  1899. 

§  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  etc.,  May  26,  i9oo,Bd.xxvn,  Nos.  18, 
19,  p.  684. 


56  Biology  of  Micro-organisms 

development  of  the  frozen  cultures  takes  place  from  the  few  surviving 
organisms. 

Bacteria  usually  grow  best  at  the  temperature  of  a  comfortably 
heated  room  (i7°C.),  and  are  not  affected  by  its  occasional  slight 
variations.  Some,  chiefly  the  pathogenic  forms,  are  not  cultivable 
except  at  the  temperature  of  the  body  (37°C.);  others,  like  the  tu- 
bercle bacillus,  grow  best  at  a  temperature  a  little  above  that  of  the 
normal  body. 

The  temperature  endurance  of  the  molds  resembles  that  of  the 
bacteria.  The  mycelia  are  killed  at  temperatures  of  6o°C.  and  over, 
but  their  spores  endure  ioo°C.  The  yeasts  and  o'idia,  that  have  no 
resisting  spores,  are  killed  at  about  6o°C.  The  protozoa  are  still 
more  sensitive  to  heat  variations  than  the  plant  organisms  and  are 
killed  by  less  extreme  variations.  Here  again,  however,  the  encysted 
protozoa  endure  greater  variations  than  the  active  organisms. 

Effect  of  Chemic  Agents. — The  presence  of  chemic  agents,  espe- 
cially certain  of  the  mineral  salts,  in  an  otherwise  perfectly  suitable 
medium  may  completely  inhibit  the  development  of  bacteria,  and 
if  added  to  grown  cultures  in  greater  concentration,  destroy  them. 
Such  substances  are  spoken  of  as  antiseptics  in  the  former,  germi- 
cides in  the  latter  case.  Bichlorid  of  mercury  and  carbolic  acid  are 
the  most  familiar  examples  of  germicides. 

Though  these  agents  are  supposed  to  operate  in  definite  concentra- 
tions with  almost  unvarying  result,  Trambusti*  found  it  possible  to 
produce  a  tolerance  to  a  certain  amount  of  bichlorid  of  mercury  by 
cultivating  Friedlander's  bacillus  upon  culture-media  containing 
gradually  increasing  amounts  of  the  salt,  until  from  1-15,000,  which 
inhibit  ordinary  cultures,  it  could  accommodate  itself  to  1-2000. 

The  various  chemic  agents  act  in  different  ways  upon  the  micro- 
organisms. Thus,  they  may  combine  with  the  protoplasm  to  make 
a  new  and  no  longer  vital  compound;  or,  they  may  coagulate  or 
dissolve  or  dehydrate  or  oxidize  the  protoplasm  to  a  destructive 
extent. 

The  addition  of  chemic  agents  to  solutions  containing  micro- 
organisms also  changes  the  osmotic  pressure.  When  an  active 
organism  is  living  in  its  normal  environment,  it  contains  within  its 
plasm  a  greater  concentration  of  solutes  than  are  to  be  found  in 
the  surrounding  fluid.  Under  these  circumstances  the  pressure 
on  the  inside  of  the  ectosarc  or  other  cell  membrane  is  greater  than 
that  on  the  outer  side,  and  the  cell  is  in  a  state  of  turgor.  If  now  salts 
are  added  so  that  the  solutes  on  the  outside  exceed  those  on  the 
inside,  water  is  drawn  out  and  the  protoplasm  is  made  to  shrink  or 
condense.  According  to  the  degree  of  this  change  the  organism 
will  be  embarrassed,  made  impotent,  or  destroyed. 

On  the  other  hand,  when  micro-organisms  have  enjoyed  a  con- 
centrated medium  like  blood-serum  and  are  suddenly  transferred  to 
*  "Lo  Sperimentale,"  1893-94. 


Fermentation  57 

distilled  water,  so  much  water  may  be  suddenly  drawn  into  their 
protoplasm  that  they  swell  up  and  may  burst  and  go  to  pieces.  This 
is  particularly  true  of  the  delicate  protozoa  like  the  trypanosoma. 

Metabolism. — According  to  their  activities,  micro-organisms  are 
classed  as — 

Zymogens,  when  they  cause  fermentation. 

Saprogens,  when  they  cause  putrefaction. 

Chromogens,  when  they  produce  colors. 

Photogens,  when  they  phosphoresce. 

Aerogens,  when  they  evolve  gas. 

Pathogens,  when  they  cause  disease. 

The  metabolic  activities  of  micro-organisms  occasion  many  well- 
known  changes  in  nature.  Thus,  it  is  through  their  energies  that  by 
fermentative  and  putrefactive  changes  organic  matter  is  gradually 
transformed  from  complex  to  simple  compounds.  It  is  by  the 
energy  of  bacteria  that  foul  waters  are  gradually  purified,  and  while 
it  is  true  that  the  presence  of  large  numbers  of  bacteria  in  water 
detracts  from  its  potability,  the  very  bacteria  that  cause  its  con- 
demnation ultimately  effect  its  purification  by  exhausting  the 
organic  matter  it  contains  in  their  own  nutrition.  In  the  treat- 
ment of  sewage  by  the  "septic  tank"  method,  the  organic  matter 
contained  in  the  water  is  consumed  through  the  agency  of  anaerobic 
and  aerobic  bacteria,  until  the  water  once  more  becomes  clear  and 
pure,  the  bacteria  dying  out  as  the  nutrition  becomes  exhausted. 

The  promptness  with  which  bacteria  attack  organic  matter  is 
seen  in  the  changes  brought  about  in  foods,  some  of  which  are 
ruined  in  flavor  or  quality,  though  others  are  thought  to  be  improved. 
Thus,  the  flavor  of  butter,  sausage,  and  cheese,  the  aroma  of  wines, 
and  many  other  important  gustatory  characteristics  of  our  foods 
depend  solely  upon  the  activity  of  bacteria  or  other  micro-organisms. 
Many  of  these  activities  are  harmless,  and,  indeed,  advantageous, 
though  the  fact  that  they  are  not  infrequently  accompanied  by 
chemic  changes,  some  of  which  are  poisonous,  makes  it  necessary  to 
watch  and  time  their  operations  lest  acridity,  acidity,  insipidity,  or 
toxicity  of  the  food  replace  the  desired  effect. 

Briefly  considered,  the  best  known  phenomena  resulting  from 
micro-organismal  energy  are  as  follows: 

Fermentation. — Fermentation  is  catalysis  of  carbon  compounds 
caused  by  catalysts  or  ferments  resulting  from  micro-organismal 
metabolism.  The  alcoholic  fermentation,  which  is  a  familiar 
phenomenon  to  the  layman  as  well  as  to  the  brewer  and  chemist, 
depends  upon  the  activity  of  an  yeast-plant,  one  of  the  saccharo- 
myces  fungi  by  which  the  sugar  is  broken  up  into  alcohol  and  carbon 
dioxid,  with  some  glycerin  and  other  by-products.  The  following 
equation  shows  the  chief  changes  produced: 

C6H12O6      =      2C2H6OH       +       2CO2 

Sugar  Alcohol  Carbon  dioxid 


58  Biology  of  Micro-organisms 

There  are  also  several  bacteria  which  produce  the  acetic  fermenta- 
tion, though  it  is  generally  attributed  to  Bacillus  aceticus.  There 
are  two  equations  to  express  this  fermentation: 

I.  CH2CH2OH      +      O      =      CH3CHO      +      H2O 

Alcohol  Oxygen   •  Aldehyd  Water 

II.  CH3CHO  +     O      =      CH3COOH 

Aldehyd  Oxygen  Acetic  acid 

A  number  of  different  bacilli  seem  capable  of  converting  milk-sugar 
into  lactic  acid,  though  Bacillus  acidi  lactici  is  the  best  known  and 
most  active  acid  producer.  The  butyric  fermentation  generally  due 
to  Bacillus  butyricus  may  also  be  caused  by  other  bacilli.  (For  an 
exact  description  of  the  chemistry  of  the  fermentations  reference 
must  be  made  to  special  text-books.  *)  The  lactic  acid  and  butyric 
acid  fermentation,  have  the  following  equations: 

I.  Ci2H22Ou       +      H20      =      CeH^O,      +      C6H1206 


Lactose  or  milk  sugar  Galactose  Dextrose 

II.     CeHiaOe       =    2C.H.O, 
Galactose  Lactic  acid 

III.       C6H1206     =       C4H802      +      C02      +      2H2 

Galactose  Butyric  acid 

Putrefaction.  —  Putrefaction  is  a  catalysis  of  proteins  resulting 
from  the  activity  of  micro-organismal  catalysts  or  enzymes.  It  is 
associated  with  the  evolution  of  a  vile  odor.  The  first  step  in  the 
process  seems  to  be  the  transformation  of  the  albumins  into  peptones, 
then  the  splitting  up  of  the  peptones  into  gases,  amino-acids,  bases, 
and  salts.  In  the  process  innocuous  albumins  are  frequently 
changed  to  toxalbumins,  and  sometimes  to  peculiar  putrefactive 
alkaloids  known  as  ptomains. 

Vaughan  and  Novy  define  a  ptomain  as  "a  chemical  compound, 
basic  in  character,  formed  by  the  action  of  bacteria  on  organic  matter  " 
The  chemistry  of  these  bodies  is  very  complex,  and  for  a  satisfac- 
tory description  of  them  Vaughan  and  Novy's  bookf  is  excellent. 

Ptomains  probably  play  but  a  small  part  in  pathologic  conditions. 
They  are  formed  almost  exclusively  outside  of  the  living  body,  and 
only  become  a  source  of  danger  when  ingested  with  the  food.  It  is 
supposed  that  cases  of  ice-cream  and  cheese  poisoning  are  usually 
due  to  tyrotoxicon,  a  ptomain  produced  by  the  putrefaction  of  the 
protein  substances  of  the  milk  before  it  is  frozen  into  ice-cream  or 
made  into  cheese.  The  safeguard  is  to  freeze  the  milk  only  when 
perfectly  fresh  and  avoid  mixing  the  milk,  cream,  sugar,  and  flavor- 
ing substances,  and  allowing  the  mixture  to  stand  for  some  time 
beforehand. 

*  See  "Enzymes  and  Their  Applications,"  by  Jean  Effront,  translated  by 
S.  C.  Prescott,  New  York,  1902;  "Micro-organisms  and  Fermentation,"  by 
Alfred  Jorgensen,  translated  by  A.  K.  Miller  and  A.  E.  Lennholm,  London, 
1900;  and  the  many  writings  of  Christian  Hansen. 

f"  Ptomaines  and  Leucomaines,"  1888;  "Cellular  Toxins,"  1902. 


Production  of  Gases 


59 


The  occasional  cases  of  "Fleischvergiftung,"  "  meat-poisoning," 
or  "Botulismus,"  are  due  to  the  development  of  toxic  ptomains  in 
consequence  of  the  growth  of  certain  bacteria  (Bacillus  botulinus)  in 
the  meat.  Kaensche*  has  carefully  investigated  the  subject,  and 
given  a  synoptic  table  containing  all  the  described  bacteria  of  this 
class.  His  researches  show  that  there  are  at  least  three  different 
bacilli  whose  growth  causes  the  meat  to  become  poisonous. 

With  the  increase  of  knowledge  upon  the  toxic  character  of  the 
bacteria  themselves,  the  importance  of  the  toxic  ptomains  has 
diminished,  until  at  present  we  have  come  to  regard  them  as  very  rare 
causes  of  disease. 

Production  of  Gases. — Various  gases  are  given  off  during  decom- 
position and  fermentation,  among  them  being  CO2,  H^S, 
H,  CH4.  Gases  produced  by  aerobic  bacteria 
usually  fly  off  from  the  surface  of  the 
culture  unnoticed,  but  if  the  bacterium 
be  anaerobic  and  develop  the  lower  part  of 
a  tube  of  solid  culture  media,  a  visible  bubble 
of  gas  is  usually  formed  about  the  colonies. 
Such  gas  bubbles  are  almost  invariably  pres- 
ent in  cultures  of  the  bacilli  of  tetanus  and 
malignant  edema. 

To  quantitatively  determine  the  gas-produc- 
tion, some  form  of  the  Smith  fermentation-tube 
is  most  convenient.  The  tube  is  filled  with 
bouillon  containing  some  sugar,  sterilized  as 
usual,  inoculated,  and  stood  aside  to  grow. 
As  the  gases  form,  the  bubbles  ascend  and 
accumulate  in  the  closed  arm.  In  estimating 
quantitatively,  one  must  be  careful  that  the 
tube  is  not  so  constructed  as  to  allow  the  gas  to 

escape  as  well  as  to  ascend   into    the   main    FiS-  16.— Smith's  fcr- 

.  mentation -tube, 

reservoir. 

For  the  determination  of  the  nature  of  the  gases  produced,  Theobald 
Smith  has  recommended  the  following  method: 

"The  bulb  is  completely  filled  with  a  2  per  cent,  solution  of  sodium  hydroxid 
(NaOH)  and  tightly  closed  with  the  thumb.  The  fluid  is  shaken  thoroughly 
with  the  gas  and  allowed  to  flow  back  and  forth  from  the  bulb  to  the  closed 
branch,  and  the  reverse  several  times  to  insure  intimate  contact  of  the  CO2 
with  the  alkali.  Lastly,  before  removing  the  thumb  all  the  gas  is  allowed  to 
collect  in  the  closed  branch  so  that  none  may  escape  when  the  thumb  is  removed. 
If  CO2  be  present,  a  partial  vacuum  in  the  closed  branch  causes  the  fluid  to  rise 
suddenly  when  the  thumb  is  removed.  After  allowing  the  layer  of  foam  to 
subside  somewhat  the  space  occupied  by  gas  is  again  measured,  and  the  difference 
between  this  amount  and  that  measured  before  shaking  with  the  sodium 
hydroxid  solution  gives  the  proportion  of  CO2  absorbed.  The  explosive  character 
of  the  residue  is  determined  as  follows:  The  cotton  plug  is  replaced  and  the 
gas  from  the  closed  branch  is  allowed  to  flow  into  the  bulb  and  mix  with  the 
air  there  present.  The  plug  is  then  removed  and  a  lighted  match  inserted  into 

*  "Zeitschrift  fur  Hygiene,"  etc.,  June  25,  1896,  Bd.  xxn,  Heft  I. 


60  Biology  of  Micro-organisms 

the  mouth  of  the  bulb.  The  intensity  of  the  explosion  varies  with  the  amount 
of  air  present  in  the  bulb.  The  relative  proportion  of  gases  resulting  from  the 
fermentation  is  frequently  of  importance  for  the  differential  diagnosis  of  related 

TT 

bacteria.     Smith  has  designated   this  relation  of  -^-   as  the   'gas    formula.' 

LU2 

The  colon  bacillus  has  a  gas  formula  corresponding  to  —^-  =  -,  Other  aerogenic 

LO2       i 

bacilli  sometimes  show  a  formula  7^-  =  -." 

LOz          2 

Liquefaction  of  Gelatin. — As  certain  organisms  grow  in  gelatin, 
the  medium  becomes  partly  or  entirely  liquefied.  This  peculiarity 
is  apparently  independent  of  any  other  property  of  the  organisms, 
and  is  manifested  alike  by  pathogenic  and  non-pathogenic  forms. 
The  liquefaction  is  supposed  to  be  dependent  upon  a  form  of  pepto- 
nization.  Bitter*  and  Sternbergf  have  shown  that  if  from  a  culture 
in  which  liquefaction  has  taken  place  the  bacteria  be  removed  by 
filtration,  the  filtrate  will  retain  the  power  of  liquefying  gelatin, 
showing  the  property  is  not  resident  in  the  bacteria,  -but  in  some 
substance  in  solution  in  their  excreted  products.  These  products  were 
described  as  "tryptic  enzymes"  by  Fermi, f  who  found  that  heat  de- 
stroyed them.  Mineral  acids  seem  to  check  their  power  to  act  upon 
gelatin.  Formalin  renders  the  gelatin  insoluble.  Some  of  the 
bacteria  liquefy  the  gelatin  in  such  a  peculiar  and  characteristic 
manner  as  to  make  the  appearance  a  valuable  guide  for  the  differen- 
tiation of  species. 

Production  of  Acids  and  Alkalies. — Under  the  head  of  "  Fermen- 
tation "  the  formation  of  acetic,  lactic,  and  butyric  acids  has  been  dis- 
cussed. Formic,  propionic,  baldrianic,  palmitic,  and  margaric  acids 
also  result  from  microbic  metabolism.  As  the  acidity  progresses, 
it  impedes,  and  ultimately  completely  inhibits,  the  activity  of 
the  organisms.  The  cultivation  of  the  bacteria  in  milk  to  which 
litmus  or  lacmoid  has  been  added  is  a  convenient  method  for  de- 
tecting changes  of  reaction.  Rosolic  acid  solutions  may  also  be 
used,  the  acid  converting  the  red  into  an  orange  color.  Neutral  red 
is  also  much  employed  for  this  purpose,  the  acids  turning  it  yellow. 

The  quantitative  estimation  of  changes  in  reaction  can  be  best 
made  by  titration,  and  the  fermentation-tube  culture  can  be  employed 
for  the  purpose.  The  contents  of  the  bulb  and  branch  should  be 
shaken  together,  a  measured  quantity  withdrawn,  and  titration  with. 

-  sodium  hydroxid,  or  —  hydrochloric  acid,  performed. 

The  alkali  most  frequently  formed  by  bacterial  growth  is  ammo- 
nium, which  is  set  free  from  its  combinations,  and  either  flies  off  as  a 
gas  or  forms  new  combinations  with  acids  simultaneously  formed. 
Some  bacteria  produce  acids  only,  some  alkalies  only,  others  both 

*  "  Archiv  fur  Hygiene,"  1886,  Heft  2. 

t"  Medical  News,"  1887,  No.  14. 

j  "Centralbl.  f.  Bakt.,"  etc.,  1891,  Bd.  x,  p.  401. 


Chromogenesis  61 

acds  and  alkalies.  Both  acids  and  the  alkalies,  when  in  excess, 
serve  to  check  the  further  activity  of  the  micro-organisms. 

Chromogenesis. — Bacteria  that  produce  colored  colonies  or  impart 
color  to  the  medium  in  which  they  grow  are  called  chromogenic; 
those  producing  no  color,  non-chromo genie.  Most  chromogenic 
bacteria  are  saprophytic  and  non-pathogenic.  Some  of  the  patho- 
genic forms,  as  Staphylococcus  pyogenes  aureus,  are,  however,  color 
producers.  It  seems  more  likely  that  certain  chromogenetic  sub- 
stances unite  with  constituents  of  the  culture  medium  to  produce  the 
colors  than  that  the  bacteria  form  the  actual  pigments;  but,  as  Gale- 
otti*  has  shown,  there  are  two  kinds  of  pigment,  one  being  soluble, 
readily  saturating  the  culture  medium,  as  the  pyocyanin  and  fluorescin 
of  Bacillus  pyocyaneus,  the  other  insoluble,  not  tingeing  the  solid 
culture  media,  but  retained  in  the  colonies,  like  the  pigment  of  Bacil- 
lus prodigiosus.  The  pigments  are  found  in  greatest  intensity  near 
the  surface  of  a  bacterial  mass.  The  coloring  matter  never  occupies 
the  cytoplasm  of  the  bacteria  (except  Bacillus  prodigiosus,  in  whose 
cells  occasional  pigment-granules  may  be  seen),  but  occurs  as  an  in- 
tercellular deposit. 

Almost  all  known  colors  are  formed  by  different  bacteria.  One 
bacterium  will  sometimes  elaborate  two  or  more  colors;  thus,  Bacillus 
pyocyaneus  produces  pyocyanin  and  fluorescin,  both  being  soluble 
pigments — one  blue,  the  other  green.  Gessardf  has  shown  that 
when  Bacillus  pyocyaneus  is  cultivated  upon  white  of  egg,  it  produces 
only  the  green  fluorescent  pigment,  but  if  cultivated  in  pure  peptone 
solution  it  produces  only  the  blue  pyocyanin.  His  experiments 
prove  the  very  interesting  fact  that  for  the  production  of  fluorescin 
it  is  necessary  that  the  culture  medium  contain  a  definite  amount 
of  a  phosphatic  salt.  Sometimes,  an  organism  produces  two  pig- 
ments, one  is  soluble,  the  other  insoluble,  so  that  the  colony  will 
appear  one  color,  the  medium  upon  which  it  grows  another.  The 
author  once  found  an  interesting  coccus,  J  with  this  peculiarity,  upon 
the  conjunctiva.  It  formed  a  brilliant  yellow  colony  upon  the  sur- 
face of  agar-agar,  but  colored  the  agar-agar  itself  a  beautiful  violet. 
In  this  case  the  yellow  pigment  was  insoluble,  the  violet  pigment 
soluble  and  diffusible  through  the  jelly.  Some  organisms  will 
only  produce  pigments  in  the  light;  others,  as  Bacillus  mycoides 
roseus,  only  in  the  dark.  Some  produce  them  only  at  the  room 
temperature,  but,  though  growing  luxuriantly  in  the  incubator,  re- 
fuse to  produce  pigments  at  so  high  a  temperature.  Thus,  Bacillus 
prodigiosus  produces  a  brilliant  red  color  when  growing  at  the  tem- 
perature of  the  room,  but  is  colorless  when  grown  in  the  incubator. 
The  reaction  of  the  culture  medium  is  also  of  much  importance  in 
this  connection.  Thus,  Bacillus  prodigiosus  produces  an  intense 

*  "Lo  Sperimentale,"  1892,  XLVI,  Fasc.  in,  p.  261. 
t"Ann.  de  PInst.  Pasteur,"  1892,  pp.  810-823. 

t  See  Norris  and  Oliver,  "  System  of  Diseases  of  the  Eye,"  vol.  H,  p.  489,  and 
"University  Medical  Magazine,"  Philadelphia,  Sept.,  1895. 


62  Biology  of  Micro-organisms 

scarlet-red  color  upon  alkaline  and  neutral  media,  but  is  colorless 
or  pinkish  upon  slightly  acid  media.  Some  of  the  pigments — per- 
haps most  of  them — are  formed  only  in  the  presence  of  oxygen. 

Production  of  Odors. — Gases,  such  as  H2S  and  NH4,  and  acids, 
butyric  and  acetic  acids,  have  sufficiently  characteristic  odors. 
There  are,  however,  a  considerable  number  of  pungent  odors  which 
seem  to  arise  from  independent  odoriferous  principles.  Many  of 
them  are  extremely  unpleasant,  as  that  of  the  tetanus  bacillus.  The 
odors  seem  to  be  peculiar  individual  characteristics  of  the  organisms. 

Production  of  Phosphorescence. — Cultures  of  Bacillus  phos- 
phorescens  and  numerous  other  organisms  are  distinctly  phosphor- 
escent. So  much  light  is  sometimes  given  out  by  gelatin  cultures 
of  these  bacteria  as  to  enable  one  to  see  the  face  of  a  watch  in  a 
dark  room.  Gorham  found  the  photogenesis  most  marked  when 
the  organisms  are  grown  in  alkaline  media  at  room  temperature. 
Most  of  the  phosphorescent  bacteria  are  found  in  sea-water,  and  are 
best  cultivated  in  sea-water  gelatin.  Some  are  familiar  to  butchers 
through  the  phosphorescence  they  cause  on  the  surface  of  stale 
meats. 

Production  of  Aromatics. — Phenol,  kresol,  hydrochinone,  hydro- 
paracumaric  acid,  and  paroxyphenylic-acetic  acid  are  by  no  means 
uncommon  products  of  bacteria.  The  most  important  is  indol, 
which  was  at  one  time  thought  to  be  peculiar  to  the  cholera  spirillum, 
but  is  now  known  to  be  produced  by  many  other  bacteria.  The 
best  method  of  testing  for  it  is  that  of  Salkowski,*  known  as  the 
nitrosoindol  reaction.  To  perform  it,  10  cc.  of  the  fluid  to  be  tested 
receive  an  addition  of  10  drops  of  concentrated  sulphuric  acid.  The 
mixture  is  shaken  in  a  test-tube.  A  few  cubic  centimeters  of  a  0.02 
per  cent,  solution  of  potassium  nitrite  are  then  allowed  to  flow  down 
the  side  of  the  tube.  If  indol  is  present,  a  purple-red  color  develops 
at  the  junction  of  the  two  fluids. f  McFarland  and  SmallJ  have 
found  that  the  intensity  of  this  color  corresponds  to  the  quantity  of 
indol  present,  and  that  quantitative  tests  can  be  made  by  means  of 
a  comparative  color  test  series. 

The  Formation  of  Nitrates. — A  process  of  fundamental  importance 
is  carried  on  by  certain  lowly  bacteria  of  the  soil.  Since  plants  are 
unable  to  assimilate  the  free  nitrogen  of  the  air,  but  must  obtain 
this  element  from  the  soil  in  the  form  of  some  soluble  compound, 
and  since  there  is  a  relatively  limited  amount  of  combined  nitrogen 
in  the  world,  it  becomes  of  the  last  importance  that  the  supplies 
which  are  continually  withdrawn .  from  the  soil  should  be  replaced 
by  the  nitrogen  liberated  in  the  decay  of  organic  material.  This 
nitrogen,  after  a  series  of  putrefactive  changes  have  occurred,  ap- 
pears as  ammonia.  The  odor  of  this  gas  is  often  plainly  perceptible 

*  "Zeitschrift.  f.  physiol.  Chemie,"  vm,  p.  417. 

f  See  Grubs  and  Francis,  "Bull,  of  the  Hyg.  Laboratory,"  1902,  No.  7. 

j  "Trans,  of  the  American  Public  Health  Association,"  1905. 


Reduction  of  Nitrates  63 

about  manure  heaps.  In  this  form  nitrogen  is  poorly  adapted  for 
use  by  plants}  and  moreover  may  be  easily  dissipated.  An  extensive 
further  process  of  oxidation  is  carried  on  by  the  nitrifying  bacteria, 
whereby  nitrates  are  ultimately  formed.  These  are  eminently 
adapted  for  use  by  plants,  and  so  the  soil  is  rendered  continuously 
capable  of  supporting  vegetation. 

Nitrosomonas  and  Nitrosococcus  convert  ammonia  into  nitrous 
acid,  and  Nitrobacter  oxidizes  the  latter  to  form  nitric  acid. 

These  genera  are  well  nigh  universal  in  the  soil.  They  do  not 
grow  on  the  ordinary  culture  media,  but  require  special  solutions, 
free  from  the  diffusive  albumins — free,  indeed,  from  organic  com- 
pounds of  any  sort.  Their  supplies  of  carbon  are  obtained  by  the 
dissociation  of  carbon  dioxid.  It  is  highly  noteworthy  that  they  are 
thus  able  to  flourish  without  food  more  complex  than  ammonia,  a 
fact  which  is  without  parallel  among  organisms  devoid  of  chlorophyl. 

Reduction  of  Nitrates. — A  considerable  number  of  bacteria  are 
able  to  reduce  nitrogen  compounds  in  the  soil  or  in  culture  media, 
prepared  for  them,  into  ammonia.  To  the  horticulturist  this  matter 
is  of  much  interest.  Winogradsky*  has  described  specific  nitrifying 
bacilli  which  he  found  in  soil,  and  asserts  that  the  presence  of  ordi- 
nary bacteria  in  the  soil  causes  no  formation  of  nitrites  so  long  as  the 
special  bacilli  are  withheld. 

Reduction  of  nitrates  can  be  determined  experimentally  by  the 
use  of  a  nitrate  broth,  made  by  dissolving  in  1000  cc.  of  water  i  gram 
of  peptone  and  0.2  gram  of  potassium  nitrate.  The  ingredients  are 
dissolved,  filtered,  then  filled  into  tubes,  and  sterilized.  The  tubes 
are  inoculated  and  the  results  noted.  As  nitrites  and  ammonia  are, 
however,  commonly  present  in  the  air  and  are  taken  up  by  fluids,  it  is 
always  well  to  control  the  test  by  an  uninoculated  tube  tested  with 
the  reagents  in  the  same  manner  as  the  culture. 

Two  solutions  are  employedf  for  testing  the  culture: 

I.  Naphthylamin,  o.i  gram,  (  Boil,  cool,    filter,    and   add    156    cc.    of 

Distilled  water,  20.0  grams,         \      dilute  (1:16)  hydric  acetate. 
II.  Sulphanilic  acid,  0.5  gram. 

Hydric  acetate,  diluted,  150.0  cc. 

Keep  the  solutions  in  glass-stoppered  bottles  and  mix  equal  parts 
for  use  at  the  time  of  employment. 

About  3  cc.  of  the  culture  and  an  equal  quantity  of  the  uninocu- 
lated culture  fluid  are  placed  in  test-tubes  and  about  2  cc.  of  the 
test  fluid  slowly  added  to  each.  The  development  of  a  red  color 
indicates  the  presence  of  nitrites,  the  intensity  of  the  color  being  in 
proportion  to  the  quantity  of  nitrites  present.  If  a  very  slight 
pinkish  or  reddish  color  in  the  uninoculated  culture  fluid  and  a  deeper 
red  in  the  culture  develop,  it  shows  that  a  small  amount  of  nitrites 


*"Ann.  de  PInst.  Pasteur,"  1891;  "La  Semaine  medicale,"  1892. 
f  "Journal  of  the  American  Public  Health  Association,"  1888,  p. 


92. 


64  Biology  of  Micro-organisms 

was  already  present,  but  that  more  have  been  produced  by  the 
growth  of  the  bacteria. 

The  presence  of  ammonia  in  either  fluid  is  easily  determined  by 
the  immediate  development  of  a  yellow  color  or  precipitate  when  a 
few  drops  of  Nessler's  solution*  are  added. 

Failure  to  determine  either  ammonia  or  nitrites  may  not  mean  that 
the  nitrates  were  not  reduced,  but  that  they  were  reduced  to  N.  It 
is,  therefore,  necessary  to  test  the  solutions  for  nitrates,  which  is 
done  by  the  use  of  phenolsulphonic  acid  and  sodium  hydroxid, 
which  in  the  presence  of  nitrates  give  a  yellow  color. 

Combination  of  Nitrogen. — Not  only  do  bacteria  destroy  or  re- 
duce nitrogen  compounds,  but  some  of  them  are  also  able  to  assimi- 
late nitrogen  from  the  air  and  so  combine  it  as  to  be  useful  for  the 
nourishment  of  vegetable  and  animal  life.  The  most  interesting 
organisms  of  this  kind  are  found  upon  the  roots  of  the  leguminous 
plants,  peas,  clover,  etc.,  and  have  been  studied  by  Beyerinck.f  It 
seems  to  be  by  the  entrance  of  these  bacteria  into  their  roots  that 
the  plants  are  able  to  assimilate  nitrogen  from  the  atmosphere  and 
enrich  sterile  ground.  Every  agriculturist  knows  how  sterile  soil  is 
improved  by  turning  under  one  or  two  crops  of  clover  with  the 
plough. 

Peptonization  of  Milk. — Numerous  bacteria  possess  the  power  of 
digesting — peptonizing — the  casein  of  milk.  The  process  varies 
with  different  bacteria,  some  digesting  the  casein  without  any  appar- 
ent change  in  the  milk,  some  producing  coagulation,  some  gelatiniza- 
tion  of  the  fluid.  In  some  cases  the  digestion  of  the  casein  is  so 
complete  as  to  transform  the  milk  into  a  transparent  watery  fluid. 

Milk  invariably  contains  large  numbers  of  bacteria,  that  enter  it 
from  the  dust  of  the  dairy,  many  of  them  possessing  this  power  and 
ultimately  spoiling  the  milk.  In  the  process  of  peptonization  the 
milk  may  become  bitter,  but  need  not  change  its  original  reaction. 

The  phenomena  of  coagulation  and  digestion  of  milk  can  be  made 
practical  use  of  to  aid  in  the  separation  of  similar  species  of  bacteria. 
Thus,  the  colon  bacillus  coagulates  milk,  but  the  typhoid  bacillus 
does  not. 

Production  of  Disease. — Micro-organisms  that  produce  disease 
are  known  as  pathogenic;  those  that  do  not,  as  non- pathogenic.  Be- 
tween the  two  groups  there  is  no  sharp  line  of  separation,  for  true 
pathogens  may  be  cultivated  under  such  adverse  conditions  that  their 
virulence  may  be  entirely  lost,  while  those  ordinarily  harmless 
may  be  made  virulent  by  certain  manipulations.  In  order  to 
determine  that  a  micro-organism  is  possessed  of  pathogenic 
powers,  the  committee  of  bacteriologists  of  the  American  Public 

*  Nessler's  solution  consists  of  potassium  iodid,  5  grams,  dissolved  in  hot 
water,  5  cc.  Add  mercuric  chlorid,  2.5  grams,  dissolved  in  10  cc.  of  water, 
then  to  the  mixture  add  potassium  hydrate,  16  grams,  dissolved  in  water,  40  cc. 
and  dilute  the  whole  to  1000  cc. 

f  "Centralbl.  f.  Bakt.,"  etc.,  Bd.  vn,  p.  338. 


Production  of  Enzymes  65 

Health  Association*  recommends  that:  (i)  When  a  given  form 
grows  only  at  or  below  18°  to  2o°C.,  inoculation  of  about  i  per  cent, 
of  the  body-weight  with  a  liquid  culture  seven  days  old  should  be 
made  into  the  dorsal  lymph-sac  of  a  frog.  (2)  When  a  species 
grows  at  25°C.  and  upward,  an  inoculation  should  be  made  into  the 
peritoneal  cavity  of  the  most  susceptible  (in  general)  of  warm- 
blooded animals — -i.e.,  the  mouse,  either  the  white  or  the  ordinary 
house  mouse.  The  inoculation  should  consist  of  about  i  per  cent, 
of  the  body-weight  of  the  mouse  of  a  four-  to  eight-hour  standard 
bouillon  culture,  or  a  broth  or  water  suspension  of  one  platinum 
loop  from  solid  cultures.  When  such  intraperitoneal  injection  fails, 
it  is  unlikely  that  other  methods  of  inoculation  will  be  successful 
in  causing  the  death  of  the  mouse.  If  the  inoculations  of  the  frog 
and  mouse  both  prove  negative,  the  committee  think  it  unnecessary 
to  insist  upon  any  further  tests  of  pathogenesis  as  being  requisite 
for  work  in  species  differentiation. 

Production  of  Enzymes. — Some  of  these  have  already  been  men- 
tioned as  causing  fermentation  and  putrefaction,  coagulating  milk, 
dissolving  gelatin,  etc.  There  are,  however,  others  which  have 
interesting  and  important  actions  upon  both  animal  and  vegetable 
substances. 

Emmerich  and  Lowf  observed  that  in  old  cultures  of  Bacillus 
pyocyaneus  the  bacteria  become  transformed  into  a  gelatinous  mass, 
and  were  led  to  experiment  with  old  and  degenerating  cultures  con- 
densed to  Mo  volume  in  a  vacuum  apparatus.  The  bacteriolytic 
powers  were  then  found  to  be  much  increased,  and  they  were  sub- 
sequently able  to  precipitate  from  the  concentrated  culture  an 
enzyme,  which  they  called  pyocyanase.  The  authors  reached  the 
rather  hasty  conclusions  that  the  cessation  of  growth  of  bacteria  in 
cultures  depends  upon  the  generation  of  enzymes;  that  the  enzymes 
destroy  the  dead  bacteria;  that  the  enzymes  will  kill  and  dissolve 
living  bacteria  and  destroy  toxins,  and,  therefore,  are  useful  for 
the  treatment  of  infectious  diseases,  and  that  antitoxins  are  simply 
accumulated  enzymes  which  the  immunized  animals  have  received 
during  treatment,  and  which,  appearing  in  the  serum,  produce 
the  effects  so  well  known. 

It  is  probable  that  many  of  the  toxic  effects  of  bacteria  and  their 
cultures  depend  upon  enzymic  substances,  the  nature  of  which  we 
do  not  yet  understand. 

*  "Jour.  Amer.  Public  Health  Assoc.,"  Jan.,  1898. 
f  "Zeitschrift  fur  Hygiene,"  1899. 


CHAPTER  III 
INFECTION 

INFECTION  is  the  successful  invasion  of  an  organism  by  micro- 
parasites.  Unfortunately  custom  has  sanctioned  the  use  of  the 
word  in  other  and  sometimes  confusing  senses,  thus,  a  table  or  knife 
upon  which  micro-organisms  are  known  to  be  or  are  even  supposed 
to  be;  the  mouth  and  intestine,  which  naturally  harbor  bacteria  of 
various  forms,  or  a  splinter  penetrating  the  skin  and  carrying  harm- 
less bacteria  into  the  deeper  tissues,  are  all  said  by  the  surgeon  to  be 
"infected"  when,  in  fact,  it  would  be  more  correct  to  describe  them 
as  infective. 

The  term  infection  should  imply  an  abnormal  state  resulting  from 
the  deleterious  action  of  the  parasite  upon  the  host.  The  colon 
bacillus  is  a  harmless  commensal  of  the  intestine  of  every  human 
being,  and  of  most  of  the  lower  animals.  The  intestine  is  not  "  in- 
fected," but  infested  with  it,  and  it  is  only  when  abnormal  or  un- 
natural conditions  arise  that  infection  can  take  place.  This  form 
of  association  of  certain  bacteria  with  certain  parts  of  the  body  to 
which  they  do  no  harm,  but  into  which  they  may  rapidly  invade 
when  appropriate  conditions  arise,  is  described  by  Adami  as  sub- 
infection.  The  possibility  of  infection  is  always  there,  though  it 
is  but  rarely  that  conditions  arise  under  which  it  can  be  accomplished. 

There  are  two  inseparable  factors  to  be  considered  in  all  infections: 
the  organism  infecting  and  the  organism  infected.  The  first  is  the 
parasite,  the  second,  the  host.  Infectivity  and  infectability  may 
depend  upon  peculiarities  of  either  parasite  or  host.  Organisms 
that  have  lived  together  as  commensals,  that  is,  in  a  state  of  neutral 
relationship  for  an  almost  indefinite  period,  may  suddenly  cease 
their  customary  association,  because  of  newly  acquired  power  of 
invasion  on  the  one  hand,  or  diminished  vital  resistance  on  the 
other,  and  infection  take  place  where  it  had  previously  been 
impossible. 

Bacteria  are  commonly  called  saprophytic  when  they  live  in  nature 
apart  from  other  living  organisms,  and  parasitic  when  they  live  in  or 
upon  them.  Saprophytic  bacteria  when  accidentally  transplanted 
from  their  natural  environment  to  the  body  of  some  animal,  for 
example,  may  or  may  not  be  capable  of  continuing  life  under  the  new 
conditions.  In  the  greater  number  of  cases  they  die,  but  sometimes 
the  new  environment  seems  better  than  the  old,  and  they  multi- 
ply rapidly,  invade  the  tissues  in  all  directions,  eliminate  their  met- 

66 


Sources  of  Infection  67 

abolic  products  into  the  juices,  and  occasion  varying  morbid 
conditions. 

The  parasitic  bacteria  live  in  habitual  association  with  higher  or- 
ganisms. Sometimes,  and  indeed  most  commonly,  it  is  a  harmless 
association,  like  that  of  certain  cocci  upon  the  skin,  but  occasionally 
it  results  in  the  destruction  of  the  tissues  and  the  death  of  the  host,  as 
in  tuberculosis,  leprosy,  etc. 

The  group  of  pathogenic  organisms  has  no  well-defined  limits,  for 
it  is  frequently  observed  that  micro-organisms  well  known  under 
other  conditions,  and  not  known  to  have  been  engaged  in  pathogenic 
processes,  turn  up  unexpectedly  as  the  cause  of  some  morbid  condi- 
tion. Indeed,  although  we  are  acquainted  with  a  large  number  of 
organisms  that  have  never  been  observed  in  connection  with  disease, 
we  are  scarcely  justified  in  concluding  that  they  are  incapable  of 
producing  injury  should  proper  conditions  arise. 

SOURCES  OF  INFECTION 

The  sources  of  infection  may  be  exogenous  or  endogenous;  that  is, 
they  may  arise  through  the  admission  to  the  tissues  of  micro-organ- 
isms from  sources  entirely  apart  from  the  individual  infected,  or 
through  the  admission  of  some  of  those  parasitic  and  usually  harmless 
organisms  constantly  associated  with  him. 

Exogenous  infections  arise  through  accidental  contact  with 
infective  agents  belonging  to  the  external  world. 

A  polluted  atmosphere  may  carry  into  the  respiratory  passages 
micro-organisms  capable  of  colonizing  there.  From  the  respiratory 
passages,  minute  drops  of  secretion  may  be  coughed  or  sneezed  into 
the  atmosphere  to  be  inhaled  by  neighboring  persons  and  infect 
them.  Such  "drop  infection"  has  been  studied  in  reference  to 
tuberculosis  and  diphtheria,  and  doubtless  explains  the  transmission 
of  whooping-cough,  pneumonia,  and  other  respiratory  disturbances. 
Polluted  water  or  food  may  carry  into  the  intestine  micro-organisms 
whose  temporary  residence  may  entirely  change  the  functional  and 
structural  integrity  of  the  parts,  as  in  typhoid  fever,  cholera  and 
dysentery. 

Wounds  inflicted  by  the  teeth  of  animals,  by  weapons,  by  imple- 
ments, or  by  objects  of  various  kinds,  carry  into  the  tissues  micro- 
organisms whose  operations,  local  or  general,  may  variously  affect 
the  organism  to  its  detriment.  Examples  are  to  be  found  in  rabies, 
tetanus,  anthrax,  malignant  and  gaseous  cedema,  suppuration,  etc. 

Fomites,  or  objects  made  infective  through  contact  with  individu- 
als suffering  from  smallpox,  scarlatina,  and  other  contagious  or 
actively  infectious  diseases,  become  the  means  through  which  the 
specific  micro-organisms  may  be  conveyed  to  the  well  with  resulting 
infection. 

Contact  with  unclean  objects  of  various  kinds — spoons,  knives,  cups, 


68  Infection 

blow-pipes,  catheters,  syringes,  dental  instruments,  etc. — may  serve 
to  transfer  disease-producing  organisms  from  one  person  to  another 
who  might  otherwise  never  come  in  contact  with  them. 

Attention  should  be  called  to  the  facility  with  which  the  diseases 
of  childhood  may  be  spread  through  the  thoughtless  or  ignorant 
custom  of  many  adults  and  children  of  using  handkerchiefs,  napkins, 
forks,  cups,  spoons,  etc.,  in  common;  in  having  wash-rags,  towels, 
hair-brushes  and  combs  in  common;  cultivating  the  habit  of  putting 
lead-pencils,  etc.,  in  the  mouth,  and  then  passing  them  on  to  others 
who  will  do  the  same,  and  to  many  other  relations  of  every-day  life  by 
which  infectious  agents  may  be  spread.  Scarlatina,  measles,  mumps, 
acute  anterior  poliomyelitis,  ophthalmia,  tuberculosis,  ringworm, 
fevers,  syphilis,  etc.,  may  all  be  spread  through  such  means. 

Suctorial  insects  seem  occasionally  to  act  as  the  medium  by  which 
micro-organisms  withdrawn  in  blood  from  one  person  may  be  in- 
troduced into  other  persons  so  that  they  become  infected.  The  flea 
thus  brings  about  the  spread  of  plague;  the  mosquito,  of  malaria;  the 
tsetse  fly,  of  trypanosomiasis;  the  tick,  of  relapsing  fever,  the  louse  of 
typhus  fever,  etc. 

Endogenous  infections  arise  through  the  activity  of  micro-organ- 
isms habitual  to  the  body.  They  indicate  morbid  conditions  of  the 
body  by  which  the  defensive  mechanisms  are  disturbed,  so  that  or- 
ganisms harmless  under  normal  conditions  become  invasive. 

All  normal  animals  are  presumably  born  free  of  parasitic  micro- 
organisms, but  it  is  impossible  for  them  to  remain  so  because  of  the 
universal  distribution  of  micro-organismal  life.  The  air,  the  water, 
the  soil,  and  the  food,  as  well  as  the  associates  of  the  young  animal, 
all  act  as  means  by  which  micro-organisms,  and  especially  bacteria, 
are  brought  to  the  surface  and  cavities  of  its  body,  and  but  a  short 
time  elapses  after  birth  before  it  harbors  the  customary  commensal 
and  parasitic  forms. 

BACTERIAL   TENANTS   OF   THE   NORMAL   HUMAN   BODY 

The  Skin  and  Adjacent  Mucous  Membranes. — The  slightly  moist 
warm  surface  of  the  skin  is  well  adapted  to  bacterial  life,  and  its  un- 
avoidable contact  with  surrounding  objects  determines  that  a  variety 
of  organisms  shall  adhere  to  it.  Of  these,  we  can  differentiate  be- 
tween forms  whose  presence  is  unexpected  and  temporary;  others 
whose  presence  may  be  expected;  and  still  others  whose  presence 
is  invariable. 

Elaborate  investigations  upon  the  bacterial  flora  of  the  skin  have 
been  made  by  Unna;*  Mittman,f  who  studied  the  finger-nails,  under 
which  he  found  no  less  than  seventy-eight  different  species ;  Maggiora,  \ 

*  "Monatshefte  fur  prakt.  Dermatol.,"  1888,  vn,  p.  817;  1889,  vm,  pp.  293, 
562;  i889^ix,  p.  49;  1890,  x,  p.  485;  1890,  xi,  p.  471;  1891,  xii,  p.  249. 
f"Archiv.  f.  path.  Anat.  u.  Phys.  u.  f.  klin.  Med.,"  1888,  cxin,  p.  203. 
j"Giornale  della  R.  Societd  d'Igiena,"  1889,  Fasc.  5,  p.  335. 


Bacterial  Tenants  of  the  Human  Body  69 

who  isolated  twenty-nine  forms  from  the  skin  of  the  foot;  and  Prein- 
delsberger,*  who  found  eighty  species  of  bacteria  on  the  hands. 
Undoubtedly  many  of  these  organisms  were  accidentally  present,  and 
were  at  least  only  semi-parasitic.  Not  a  few  were  met  but  once 
and  were  in  no  sense  bacteria  of  the  skin.  The  skin  may  also  be 
temporarily  contaminated  with  bacteria  from  other  portions  of  the 
patient's  body,  as,  for  instance,  from  his  intestine;  thus  Winslowf 
has  found  the  colon  bacillus  upon  the  hands  of  ten  out  of  one  hundred 
and  eleven  persons  examined.  Wiguraf  also  examined  the  hands 
of  forty  persons  in  hospitals,  finding  tubercle  bacilli  in  two  out  of 
ten  persons  from  phthisical  wards,  colon  bacilli  six  times  and  typhoid 
bacilli  once  on  the  hands  of  nine  attendants  in  the  typhoid  wards. 
He  found  streptococci  and  staphylococci  many  times.  Welch§  and 
Robb  and  Ghriskey||  seem  to  have  been  the  first  to  make  a  clear 
differentiation  between  the  accidentally  present  bacteria  and  the 
permanently  parasitic  organisms  of  the  skin,  and  to  show  that  cer- 
tain cocci,  producing  white  and  yellow  colonies  upon  agar-agar, 
were  invariable  in  occurrence  and  penetrated  to  the  lowest  epidermal 
layers. 

These  cocci,  of  which  .  Welch  describes  the  most  common  as 
Staphylococcus  epidermidis  albus,  are  universally  and  invariably 
present  upon  the  human  skin,  and  must  be  regarded  as  habitual 
parasites. 

Where  the  skin  is  peculiar  in  its  moisture  and  greasiness,  however, 
additional  forms  are  found.  Thus,  in  preputial  smegma,  in  the  axillae, 
and  sometimes  about  the  lips  and  nostrils,  a  bacillary  organism, 
Bacillus  smegmatis,  is  invariable,  and  the  recent  work  by  Schaudinn 
and  Hoffmann**  has  shown  that  the  skin  of  the  genitalia  harbors 
a  spiral  organism  which  they  call  Spirochaeta  refringens. 

In  the  external  auditory  meatus  a  coccus,  Micrococcus  cereus  flavus, 
is  almost  always  to  be  found  in  the  waxy  secretion. 

Upon  the  conjunctiva  as  many  accidental  organisms  may  be  found 
as  shall  have  been  caught  by  its  moist  surface,  though  the  researches 
of  Hildebrand  and  Bernheim  and  others  seem  to  show  that  the  tears 
have  some  antiseptic  power  and  prevent  the  organisms  from  growing, 
so  that  in  health  there  are  very  few  permanent  residents  of  the  sac, 
certain  cocci  seeming  to  be  the  only  constant  forms. 

The  mouth  has  been  carefully  studied  bacteriologically  by  Miller,  ft 
who  found  six  organisms— Leptothrix  innominata,  Bacillus  buccalis 

*  "Samml.  medic.  Schriften,"  herausg.  von  der  "Wiener  klin.  Wochenschrift," 
1891;  xxn,  Wien,  "Rev.  Jahresbericht  uber  die  Fortschritten  in  der  Lehre  von 
den  pathogenen  Mikroorganismen,"  1891,  vn,  p.  619. 

"Jour.  Med.  Research,"  vol.  x,  p.  463. 
f"Wratsch,"  1895,    No.  14. 

"Transactions  of  the  Congress  of  American    Physicians   and    Surgeons," 
,  H,  p.  i. 

"Bulletin  of  the  Johns  Hopkins  Hospital,"  1892,  in,  p.  37- 
'*  "Deutsche  med.  Woch.,"  May  5,  1905. 
ft" Micro-organisms  of  the  Human  Mouth,"  Phila.,  1890. 


70  Infection 

maximus,  Leptothrix  buccalis  maxima,  lodococcus  vaginatus,  Spiril- 
lum sputigenum  and  Spirochaeta  dentinum  (denticola) — in  every 
mouth.  Practically  the  same  conclusions  were  reached  by  Vin- 
centini.*  These  organisms  are  peculiar  in  that  they  will  not  grow  in 
artificial  culture.  In  addition  to  this  permanent  flora,  Miller  culti- 
vated fifty- two  other  species,  some  of  which  were  harmless,  some  well- 
known  pathogens. 

From  the  mouth  these  organisms  may  be  traced  into  the  pharynx 
and  esophagus. 

In  studying  the  micro-organisms  of  dental  caries  Goodbyf  found 
a  large  number  of  organisms  which  he  divided  into  three  groups: 
A.  Those  that  produce  acids,  including  Streptococcus  brevis,  Ba- 
cillus necrodentalis  (Goodby),  Sarcina  alba,  Sarcina  lutea,  Sarcina 
aurantiaca,  Staphylococcus  pyogenes  aureus,  and  Staphylococcus 
pyogenes  salivarius  (Biondi).  B.  Those  that  liquefy  blood-serum: 
Bacillus  mesentericus  rubra,  B.  mesentericus  vulgatus,  B.  mesenteri- 
cus  fuscus,  Bacillus  fuscus,  a  yellow  bacillus,  probably  B.  gingivse 
pyogenes  (Miller),  and  Bacillus  liquefacium  motilis.  C.  Those  that 
produce  pigment,  including  the  same  organisms  as  group  B.  In  ca- 
rious dentine  two  organisms,  Streptococcus  brevis  and  Bacillus 
necrodentalis,  were  invariably  present. 

The  extinction  of  the  great  number  of  bacteria  entering  the  mouth 
is  referred  by  most  bacteriologists  to  a  bactericidal  action  of  the 
saliva. 

The  stomach  seems  to  retain  very  few  of  the  many  bacteria  that 
must  enter  it,  its  persistently  acid  contents  being  inimical  to  their 
development.  Certain  sarcina,  especially  Sarcina  ventriculi,  may  be 
found  without  any  considerable  departure  from  the  normal  state. 
In  carcinoma  and  other  forms  of  pyloric  obstruction  with  dilatation, 
the  bacterial  flora  increases,  and  in  achlorhydria  micro-organisms 
of  fermentation  make  their  appearance.  They  are,  however,  acci- 
dental and  not  permanent  tenants  of  the  organ. 

In  carcinoma  of  the  stomach  a  bacillus,  probably  one  of  the  lactic 
acid  groups,  early  makes  its  appearance  and  is  of  some  diagnostic  im- 
portance. It  is  called  after  its  discoverer  the  Oppler-Boas  bacillus,  J 
also  on  account  of  angulations  found  in  its  threads,  Bacillus  gen- 
iculatus.  It  is  a  large  bacillus,  tending  to  form  long  threads  easily 
seen  without  an  oil-immersion  lens.  It  is  probably  non-motile,  does 
not  form  spores,  stains  by  Gram's  method,  and  is  said  by  Emory§ 
to  divide  longitudinally  as  well  as  transversely.  This,  as  he  says,  will, 
if  proved  to  be  correct,  be  a  most  important  means  of  identifying  the 
species.  Cultures  are  easily  made  in  media  acidified  with  lactic  acid. 

The  intestine  receives  such  micro-organisms  as  have  survived  what- 
ever destructive  influences  the  gastric  juices  may  have  exerted,  and 

*  "  Bacteria  of  the  Sputa  and  Cryptogamic  Flora  of  the  Mouth,"  London,  1897- 
f  Transactions  of  the  Odontological  Society,  June,  1899. 
j  "  Deutsche  med.  Wochenschrift,"  1905,  No.  5. 
§  "Bacteriology  and  Hematology,"  p.  114. 


Bacterial  Tenants  of  the  Normal  Human  Body         71 

its  alkaline  contents,  rich  in  proteins  and  carbohydrates  in  solution, 
are  eminently  appropriate  for  bacterial  life.  The  flora  of  the  intes- 
tine is,  therefore,  increased  in  number  and  variety  of  organisms  as  we 
descend  from  its  beginning  to  its  end.  In  the  small  intestine  there 
may  be  no  bacteria  in  the  upper  part  of  the  jejunum,  but  in  most  cases 
Bacillus  lactis  aerogenes  and  bacilli  of  the  colon  groups  are  found. 
These  increase  in  number  as  the  iliocecal  valve  is  reached.  The 
cecum  shows  large  numbers  of  colon  bacilli.  The  rectum  con- 
tains, in  addition,  many  putrefactive  organisms,  such  as  Bacillus 
putrificus,  Bacillus  proteus  vulgaris,  members  of  the  Bacillus  subtilis 
group,  and  acid-producing  organisms,  such  as  Bacillus  acidophilus. 

An  interesting  and  thorough  study  of  these  organisms  of  the 
bowel  and  their  distribution  has  been  made  by  Kohlbrugge.*  The 
total  number  of  permanent  residents  is  not  known.  During  in- 
fancy the  predominating  organism  seems  to  be  Bacillus  ]actis 
aerogenes;  during  adult  life,  Bacillus  coli.  Streptococci,  especially 
Streptococcus  coli  gracilis,  are  also  very  common,  if  not  invariable, 
inhabitants  of  the  intestine.  The  total  bacteria  that  finally  appear 
in  the  feces,  according  to  the  studies  of  Strasburgerf  and  Steele,J 
may  reach  the  enormous  figure  of  38  per  cent,  of  the  total  bulk. 

MacNeal,  Latzer,  and  Kerr,§  in  an  elaborate  work  upon  the 
"Fecal  Bacteria  of  Healthy  Men,"  found  that  they  furnished  46.3 
per  cent,  of  the  total  fecal  nitrogen. 

Rettger||  found  the  Bacillus  enteritidis  sporogenes  regularly  pres- 
ent in  the  human  feces  and  believes  it  to  be  responsible  for  some  of 
the  putrefactive  processes  that  occur  there. 

The  vagina,  on  account  of  its  acid  secretions,  harbors  but  few 
bacteria.  In  a  study  of  the  vaginal  secretions  of  40  pregnant 
women  who  had  not  been  subjected  to  digital  examinations,  douches, 
or  baths,  Bergholm**  found  but  few  organisms  of  limited  variety. 

The  uterus  harbors  no  bacteria  in  health,  and  but  few  in  disease. 
The  intervening  acidity  of  the  vagina  makes  it  difficult  for  bacteria 
from  the  surface  to  penetrate  so  deeply,  and  the  tenacious  alkaline 
mucus  of  the  cervix  is  an  additional  barrier  to  their  progress.  Care- 
ful studies  of  the  bacteriology  of  the  uterine  secretions  have  been 
made  by  Gottschalk  and  Immerwahrtt  and  Doderlein  and 

Winterintz.Jt 

The  urethra  harbors  a  few  cocci  which  enter  the  meatus  from  the 
surface  and  remain  local  in  distribution. 

The  normal  bladder  is  free  from  bacteria. 

The  nose  constantly  receives  enormous  numbers  of  bacteria  in  the 

*  'Centralbl.  f.  Bakt.,"  etc.,  1901,  Bd.  xxx,  pp.  10  and  70. 

t  'Zeitschrift  fur  klin.  Med.,"  1902,  XLIV,  5  and  6;  1903,  XLVITI,  5  and  6. 

j  'Jour.  Amer.  Med.  Assoc.,"  Aug.  24,  1907,  p.  647. 

§  'Journal  of  Infectious  Diseases,"  1909,  vi,  pp.  132,  571. 

|j   'Jour,  of  Biological  Chemistry,"  Aug.,  1906,  n,  T  and  2,  p.  71. 
**  'Archiv  f.  Gynak.,"  Bd.  LXIV,  Heft  3. 
ft  Ibid.,  1896,  Bd.  L,  Heft  3. 
tJ"Beitrage  fur  Geburtshulfe  und  Gynakologie,"  Bd.  in,  Heft  2. 


72  Infection 

dust  of  the  inspired  atmosphere.  These  organisms  are  too  numer- 
ous and  too  various  to  enumerate,  and  might,  indeed,  comprehend 
the  entire  bacterial  flora.  But  in  spite  of  the  large  numbers  of  organ- 
isms received,  the  nose  retains  scarcely  any,  its  mucous  membranes 
seeming  to  be  provided  with  means  of  disposing  of  the  organisms. 
Among  those  best  able  to  withstand  the  destructive  influences,  and, 
therefore,  most  apt  to  be  found  in  the  deeper  passages,  are  the  pseudo- 
diphtheria  bacillus,  streptococci,  pneumococci,  staphylococci,  Ba- 
cillus pneumoniae  (Friedlander),  Bacillus  subtilis  and  sarcina.  A 
complete  review  of  the  subject  with  references  to  the  literature 
has  been  made  by  Hasslauer.* 

The  larynx  and  trachea  contain  very  few  bacteria  and  probably 
have  no  permanent  parasitic  flora. 

The  lungs  harbor  no  bacteria.  A  few  micro-organisms  doubtless 
reach  them  in  the  inspired  air,  but  the  defensive  mechanisms  soon 
dispose  of  them. 

AVENUES  OF  INFECTION 

The  skin  seems  to  form  an  effectual  barrier  against  the  entrance 
of  bacteria  into  the  deeper  tissues.  A  few  higher  fungi — Tryco- 
phyton,  Microsporon,  Achorion,  etc. — seem  able  to  establish  them- 
selves in  the  superficial  layers  of  the  cells,  invade  the  hair-follicles, 
and  so  reach  the  deeper  layers,  where  morbid  changes  are  produced. 
The  minute  size  of  the  bacteria  makes  it  possible  for  them  to  enter 
through  lesions  too  small  to  be  noticed.  Garre  applied  a  pure 
culture  of  Staphylococcus  pyogenes  aureus  to  the  skin  of  his  fore- 
arm, and  found  that  furuncles  developed  in  four  days,  though  the 
skin  was  supposed  to  be  uninjured.  Bockhart  moistened  his  skin 
with  a  suspension  of  the  same  organism,  gently  scratched  it  with 
his  finger-nail,  and  suffered  from  a  furuncle  some  days  later. 

The  greater  number  of  surgical  infections  result  from  the  entrance 
of  bacteria  through  lesions  of  the  skin.  It  makes  but  little  difference 
to  what  depth  the  lesion  extends — abrasions,  punctures,  lacerations, 
incisions — the  protective  covering  is  gone  and  the  infecting  organ- 
isms find  themselves  in  the  tissues,  surrounded  by  the  tissue  lymph, 
under  conditions  appropriate  for  growth  and  multiplication,  provided 
no  inhibiting  or  destructive  mechanism  be  called  into  action. 

The  digestive  apparatus  is  the  portal  through  which  many  infec- 
tions take  place.  The  Bacillus  diphtherias,  finding  its  way  to  the 
pharynx,  speedily  establishes  itself  upon  the  surface,  producing 
pseudomembranous  inflammation  there.  Typhoid  bacilli,  dysentery 
amoeba  and  bacilli,  cholera  spirilla  and  related  organisms,  finding  their 
way  to  the  intestine,  where  the  vital  conditions  are  appropriate,  take 
up  temporary  residence  there,  to  the  injury  of  the  host,  who  may 
suffer  from  the  respective  infections. 

*  "  Centralbl.  f .  Bakt.  u.  Parasitenk.  I.  Abt.  Referata,"  Bd.  "xxxvn,  Nos. 
1-3,  p.  i,  and  Nos.  4-6,  p.  97. 


Avenues  of  Infection  73 

Various  organisms  pass  from  the  pharynx  to  the  tonsils  and  so 
to  the  lymph-nodes  and  deeper  tissues  of  the  neck,  where  their  first 
operations  may  be  observed. 

It  is  supposed  by  some  pathologists  that  the  digestive  tract  is  a 
constant  menace  to  health  in  that  it  regularly  admits  bacteria, 
through  the  lacteals,  and  perhaps  through  its  capillaries,  to  the 
blood,  where  under  slightly  abnormal  conditions  they  might  do 
harm.  According  to  Adami,*  the  intestine  is  responsible  for  a 
condition  of  sub-infection  depending  upon  the  constant  entrance 
of  colon  bacilli  into  the  blood.  He  finds  the  colon  bacillus  in 
the  blood,  and  traces  it  to  the  liver,  where  its  final  dissolution 
takes  place  in  the  fine  dumbbell-like  granules  enclosed  in  the 
cells.  Nichollsf  confirms  Adami  by  finding  similar  dumbbell 
or  diplococcoid  bodies  in  the  epithelial  denuded  tissues  of  the 
mesentery  of  normal  animals. 

Nicholas  and  DescosJ  and  Ravenel§  fed  fasting  dogs  upon  a  soup 
containing  quantities  of  tubercle  bacilli,  killed  them  three  hours 
later,  and  examined  the  contents  of  the  thoracic  duct,  where 
tubercle  bacilli,  some  alive  and  some  dead,  were  found  in  large 
numbers,  van  Steenberghe  and  Grysez||  found  that  carbon  particles 
readily  passed  through  the  intestinal  mucosa,  entered  the  lymphatics, 
were  thrown  into  the  venous  circulation,  and  so  carried  to  the  lung, 
where  anthracosis  was  produced. 

In  a  subsequent  paper**  they  believe  that  they  have  demonstrated 
that  the  tubercle  bacillus  like  the  carbon  particles  may  also  pass 
through  the  normal  intestinal  wall,  and  follow  the  same  course  to 
the  lungs.  They  believe  that  pulmonary  tuberculosis  thus  depends 
upon  ingested  and  not  inhaled  micro-organisms.  Montgomery  1 1  re- 
pea  ted  the  work  of  van  Steenberghe  and  Grysez  at  the  Henry  Phipps 
Institute,  Philadelphia,  but  though  many  attempts  were  made  by 
various  methods,  no  carbon  particles  seemed  to  be  transported  from 
the  alimentary  to  the  pulmonary  tissues. 

But  there  are  enough  experiments  recorded  to  make  it  probable 
that  the  wall  of  the  intestine  is  permeable  to  bacteria,  and  that  in 
small  numbers  they  constantly  enter  the  blood  of  healthy  animals, 
to  be  disposed  of  by  mechanisms  yet  to  be  described. 

Many  of  the  bacteria  penetrating  the  intestine  must  be  retained 
in  the  lymph  nodes;  others,  as  in  the  experiment  with  the  tubercle 
bacilli,  meet  destruction  before  they  reach  the  blood;  the  remainder 
must  reach  the  blood  alive. 

The  presence  of  colon  bacilli  in  the  greater  number  of  the  organs 

*"Jour.  of  the  American  Medical  Association,"  Dec.  16  and  23,  1899,  vol. 
xxxm,  Nov.  25  and  26. 

t  "Jour.  Med.  Research,"  vol.  xi,  No.  2. 

J"Jour.  de  Phys.  et  Path.  ge"n.,"  1902,  iv,  910-912. 

§  "Jour.  Med.  Research,"  1904,  x,  p.  460. 

||  "Ann.  de  1'Inst.  Pasteur,"  Dec.  25,  1905,  Tome  xix,  No.  12,  p.  787. 
**  Ibid.,  1310,  xxiv,  316. 
ft  "Jour,  of  Med.  Research,"  Aug.,  1910,  vol.  xxm,  No.  i. 


74  Infection 

shortly  after  death  has  led  some  pathologists  to  assume  that  they 
readily  pass  through  the  intestinal  walls  during  the  death  agony, 
but  although  experiments  have  been  made  to  prove  and  to  disprove 
it,  the  matter  is  still  controversial.  Undoubtedly  in  the  final  dis- 
solution some  change  takes  place  in  the  constitution  of  the  individual 
by  which  general  invasion  by  bacteria  is  made  more  easy  than  under 
normal  conditions. 

The  respiratory  apparatus  affords  admission  to  a  few  micro-organ- 
isms whose  activities  seem  more  easily  carried  on  there  than  else- 
where. Although  it  is  still  controversial  whether  the  inhalation  of 
tubercle  bacilli  is  as  frequent  a  mode  of  conveying  that  organism  into 
the  body  as  was  once  supposed,  it  cannot  be  denied  that  its  inhalation 
will  account  for  the  far  greater  frequency  with  which  tuberculosis 
affects  the  lungs  than  other  organs  of  the  body. 

Pneumonia,  caused  in  an  immense  majority  of  cases  by  the  pneu- 
mococcus  of  Fraenkel  and  Weichselbaum,  probably  results  from  the 
entrance  of  the  organism  into  the  respiratory  tissues  directly. 

The  entrance  of  the  unknown  infectious  agents  causing  measles, 
German  measles,  smallpox,  and  scarlatina  can  best  be  accounted 
for  by  supposing  that  they  are  inhaled  into  the  lungs  and  thus  enter 
the  blood. 

The  genital  apparatus  is  the  portal  of  entry  of  micro-organisms 
whose  early  or  chief  operations  are  local.  Among  these  are  the 
gonococcus,  which  causes  urethritis,  vaginitis,  balanitis,  posthitis, 
endometritis,  orchitis,  salpingitis,  vesiculitis,  cystitis,  oophoritis, 
sometimes  peritonitis,  and  rarely  endocarditis;  the  bacillus  of  Ducrey, 
that  causes  the  chancroid  or  soft  sore;  and  the  treponema  of  syphilis. 
In  more  rare  cases  other  organisms,  such  as  the  common  cocci  of 
suppuration  and  the  tubercle  bacillus,  may  also  be  transmitted  from 
individual  to  individual  by  sexual  contact. 

The  placenta  usually  forms  a  barrier  through  which  infectious 
agents  find  their  way  with  difficulty.  A  study  of  this  subject  by 
Neelow*  shows  that  the  non-pathogenic  organisms  do  not  pass 
from  the  mother  through  the  placenta  to  the  fetus.  Some  patho- 
genic micro-organisms,  however,  readily  pass  through,  and  a  few 
diseases,  such  as  syphilis,  are  well  known  in  the  congenital  form. 
Pregnant  women  suffering  from  smallpox  may  be  delivered  of  in- 
fants with  marks  indicative  of  prenatal  disease.  Some  common  in- 
fectious agents,  such  as  the  tubercle  bacillus,  seem  to  infect  unborn 
animals  with  difficulty.  The  frequency  of  antenatal  tuberculous 
infection  is,  however,  somewhat  controversial  at  present,  Baum- 
garten  having  reached  the  opinion,  exactly  the  opposite  of  what 
is  commonly  believed,  that  many  children  are  subject  to  antenatal 
infection,  though  the  bacilli  subsequently  develop  and  cause  disease 
in  only  a  few  of  them. 

*  "Centralbl.  f.  Bakt.,"  etc.,  Aug.,  1902,  I.  Abt.,  Bd.  xxxi,  Orig.,  p.  691. 


Pathogenesis  75 

PATHOGENESIS 

This  subject  can  be  understood  only  through  a  broad  knowledge 
of  the  metabolic  products  of  micro-organisms.  In  general  it  may 
be  said  that  the  ability  of  micro-organisms  to  do  harm  depends  upon 
the  injurious  nature  of  their  products.  This  alone,  however,  will 
not  explain  the  phenomena  of  infection,  for  in  many  cases  the  in- 
toxication is  subsidiary  in  importance  to  the  invasive  power  of  the 
micro-organisms.  Some  bacteria  having  but  limited  toxic  powers 
possess  extraordinary  powers  of  invasion,  as  Bacillus  anthracis, 
and  the  intoxication  becomes  important  only  after  the  organisms 
have  penetrated  to  all  the  tissues  of  the  body.  Others,  with  more 
active  toxic  properties,  have  but  limited  invasive  powers,  and  a  few 
organisms,  growing  with  difficulty  in  some  insignificant  focus,  ex- 
cite actively  destructive  reactions  in  the  tissues  with  which  they 
come  in  contact.  Still  others,  with  limited  invasive  powers, 
eliminate  active  toxic  substances,  soluble  in  nature,  that  enter  the 
circulation  and  act  upon  cells  remote  from  the  bacteria  themselves, 
as  in  diphtheria  and  tetanus. 

The  invasive  power  of  the  organisms  depends  upon  their  ability 
to  overcome  the  body  defenses.  This  may  indicate  activity  of  the 
infecting  organism,  or  weakness  of  the  defensive  mechanism.  The 
relation  of  these  factors  is  exceedingly  complex,  only  partly  under- 
stood, and  will  be  fully  discussed  in  the  chapter  upon  Immunity. 

For  convenience  toxins  may  be  described  as  intracellular  or  in- 
soluble, and  extracellular  or  soluble. 

The  intracellular  toxins.  Until  the  investigations  of  Vaughan, 
Cooley  and  Gelston,*  and  later  Vaughan  and  his  associates,  Det- 
weiler,f  Wheeler, {  Leach, §  Marshall  and  Gelston, ||  Gelston,**  J. 
V.  Vaughan,ft  Wheeler, It  Leach,§§  Mclntyre,||||  and  others,  it 
seemed  remarkable  that  micro-organisms  whose  filtered  cultures 
contained  little  demonstrable  toxic  substance  are  sometimes  able 
to  produce  active  pathogenic  effects.  By  means  of  special  apparatus 
in  which  the  micro-organisms  could  be  cultivated  in  enormous  quan- 
tities, and  the  disintegration  of  the  micro-organismal  masses  secured 
by  subjecting  them  to  high  temperatures,  to  the  action  of  mineral 
acids  or  autolysis,  it  was  discovered  that  the  colon  bacilli,  typhoid 
bacilli,  and  many  supposedly  harmless  bacteria  contain  intensely 
active  toxic  substances.  In  all  probability  some  of  the  toxic  sub- 
stances produced  by  such  means  are  artefacts,  but  enough  work 
has  been  done  to  prove  that  insoluble  toxic  substances  are  present 
in  such  organisms,  and  the  toxic  substances  obtained  by  the  com- 

*" Journal  of  the  American  Medical  Association,"  Feb.  23,  1901;  "Trans. 
Assoc.  Amer.  Phys.,"  1901;  "American  Medicine,"  May,  1901. 
t    "Trans  Asso.  Amer.  Phys.,"  1902.  J  Ibid. 

§    Ibid.  ||  Ibid.  **  Ibid. 

ft  Ibid. 

ft  "Jour.  Amer.  Med.  Assoc.,"  1904,  XLII,  p.  1000. 
§§  Ibid.,  p.  1003.  III!  Ibid.,  p.  1073. 


76  Infection 

minution  of  culture  masses  made  solid  and  brittle  by  exposure  to 
liquid  air,  as  suggested  by  Macfadyen  and  Rowland;  the  autolytic 
digestion  of  bacteria  washed  free  of  their  culture  fluids  and  suspended 
in  physiological  salt  solution,  and  the  dissolution  of  bacteria  by 
bacteriolytic  animal  juices  clearly  prove  that  endotoxins  exist. 

It  seems  probable  that  there  is  considerable  difference  in  the 
readiness  with  which  these  intracellular  toxic  substances  are  given 
up  by  the  bacteria.  From  some  they  seem  never  to  be  set  free  in 
the  bodies  of  animals  into  which  the  bacteria  are  injected;  thus, 
Bacillus  prodigiosus  is  usually  harmless  for  animals,  no  matter  what 
quantity  is  injected,  yet  active  toxic  substances  can  be  extracted 
from  the  bodies  of  these  organisms  by  appropriate  chemical  means. 
From  others  they  are  given  off  in  small  quantities  either  during 
the  life  of  the  organism  or  at  the  moment  of  death  and  dissolution, 
as  in  the  case  of  the  typhoid  bacillus  and  streptococci,  whose  filtered 
cultures  are  almost  harmless,  though  both  organisms  are  pathogenic. 

The  intracellular  toxins  are  limited  in  action  by  the  distribution 
of  the  bacteria  producing  them.  When  these  organisms  are  but 
slightly  invasive,  more  or  less  local  reaction  is  produced;  when  they 
are  actively  invasive,  general  reactions  of  varying  intensity  result. 

The  extracellular  toxins,  of  which  those  of  Bacillus  tetani  and 
Bacillus  diphtheriae  can  be  taken  as  types,  have  been  known  since 
the  early  work  of  Brieger  and  Frankel  and  Roux  and  Yersin.  They 
seem  to  be  excretions  of  the  bacteria,  not  retained  in  the  cells,  but 
eliminated  from  them  as  rapidly  as  they  are  formed.  Thus,  in 
appropriate  bouillon  cultures  of  the  diphtheria  bacillus,  the  toxin 
is  present  in  large  quantity  and  is  highly  virulent,  but  if  the  fluid  be 
removed  from  the  bacteria  by  porcelain  filtration  and  the  remaining 
bacilli  carefully  washed,  their  bodies  are  found  to  be  devoid  of 
toxic  powers.  The  poison  is  most  concentrated  where  its  diffusion 
is  most  restricted,  thus,  agar-agar  cultures  of  the  tetanus  bacillus 
are  much  more  toxic  than  bouillon  cultures  because  the  soluble 
principle  readily  diffuses  through  the  fluid,  but  is  held  by  the  agar- 
agar. 

The  soluble  toxin  is  but  one  of  numerous  metabolic  products  of 
the  bacteria.  Thus  in  culture  filtrates  of  the  tetanus  bacillus  there 
are  at  least  two  very  different  active  substances,  the  tetano-spasmin 
that  acts  upon  the  nervous  system  with  convulsive  effect,  and  the 
tetano-lysin  that  is  solvent  for  erythrocytes. 

In  all  probability  all  of  the  culture  filtrates  of  bacteria  are  highly 
complex  because  of  the  addition  of  the  various  metabolic  products 
—toxins,  lysins,  enzymes,  pigments,  acids,  etc. — of  the  bacteria, 
as  well  as  because  of  changes  produced  in  the  medium  by  the  ab- 
straction of  those  molecular  constituents  upon  which  the  bacteria 
have  fed.  This  complexity  makes  it  difficult  to  accurately  study 
the  toxins,  which  we  scarcely  know  apart  from  their  associated 
products. 


Specific  Action  of  Toxins  77 

The  chemic  nature  of  the  toxins  differs.  Undoubtedly  some  are 
tox-albumins,  but  others  are  of  different  composition  and  fail  to 
give  the  reactions  belonging  to  the  compounds  of  this  group. 

The  variations  observed  in  toxicogenesis  under  experimental 
conditions  in  the  test-tube  indicate  that  similar  variations  occur 
in  the  bodies  of  animals,  and  a  few  experiments  conducted  with 
slight  variations  in  the  composition  and  reaction  of  the  media  in 
which  the  bacteria  grow  will  suffice  to  show  that  the  exact  effect 
of  toxicogenic  bacteria  in  the  bodies  of  different  animals  cannot 
always  be  accurately  prejudged. 

The  physiologic  and  pathogenic  action  of  the  extracellular  soluble 
toxins  differs  from  that  of  the  intracellular  and  difficultly  soluble 
toxins  in  that  it  is  more  easily  diffused  throughout  the  animal  juices, 
and  that  its  diffusion  is  independent  of  the  invasiveness  of  the  bac- 
teria, so  that  a  few  organisms  growing  at  some  focus  of  unimportant 
magnitude,  and  causing  but  little  local  manifestation,  may  be  able 
to  produce  a  profound  impression  upon  remote  organs.  This 
is  best  exemplified  in  the  case  of  the  Bacillus  tetani,  which,  finding 
its  way  into  the  tissues  under  proper  conditions,  produces  scarcely 
any  local  reaction — indeed,  the  lesion  may  be  undiscoverable — 
yet  may  cause  the  death  of  the  animal  through  the  intensity  of  its 
action  upon  the  central  nervous  system. 

SPECIFIC  ACTION  OF  TOXINS 

The  metabolic  products  of  the  greater  number  of  injurious  bacteria 
are  characterized  by  irritative  action  upon  those  body  cells  with 
which  they  come  into  contact.  If  through  the  intracellular  nature 
of  the  poisons  and  the  mildly  invasive  character  of  the  micro- 
organisms this  action  is  restricted  to  the  seat  of  original  infection,  a 
local  manifestation  will  result.  Its  exact  nature  will,  however,  be 
modified  to  some  extent  by  other  qualities  of  the  bacterial  products. 
Thus,  when  in  addition  to  their  irritative  action  which,  when  mild, 
occasions  multiplication  of  the  cells  of  the  connective  and  lymphoid 
tissues,  and,  when  extreme,  effects  the  death  of  the  cells,  the  products 
are  strongly  chemotactic,  suppuration  will  occur. 

Fever  and  suppuration  are,  therefore,  non-specific  actions,  because 
numerous  micro-organisms  share  in  common  the  qualities  produc- 
tive of  these  conditions. 

If  the  bacteria  are  rapidly  invasive,  but  still  have  injurious 
products  of  the  intracellular  variety,  they  are  apt  to  share  certain 
qualities,  such  as  the  swelling  of  the  lymph-nodes,  etc.,  in  common, 
so  that  such  lesions  cannot  be  considered  as  specific.  So  soon  as 
any  one  of  the  products  is  discovered  to  give  some  single  lesion 
peculiar  to  that  organism  by  which  it  is  produced,  or  so  soon  as  the 
total  effect  of  the  activity  of  the  various  products  of  any  micro- 
organism produces  a  typical  effect,  differing  from  the  total  effect 


78  Infection 

of  the  operation  of  other  micro-organisms,  and  a  recognized  type 
of  disease  results,  it  becomes  possible  to  say  that  the  micro-organism 
in  question  is  specific. 

The  most  striking  examples  of  the  specific  action  of  bacterio- 
toxins  is,  however,  seen  in  those  cases  where  soluble  extracellular 
metabolic  products  of  bacterial  energy  are  liberated  into  the  body 
juices  so  as  to  be  conveyed  by  the  circulatory  system  to  all  parts 
of  the  body.  Those  cells  most  susceptible  to  its  action  are  then 
first  or  most  profoundly  impressed  by  it,  and  definite  responses 
brought  about.  Thus,  the  soluble  toxin  of  tetanus  causes  no  visible 
reaction  in  the  cells  with  which  it  first  comes  into  contact  at  the 
seat  of  primary  infection,  because  these  cells  are  either  less  sus- 
ceptible to  its  influence,  or  are  less  well  able  to  show  its  effects, 
than  the  cells  of  the  nervous  system  to  which  it  is  secondarily  carried 
by  the  blood. 

SPECIFIC  AFFINITY  OF  THE  CELLS  FOR  THE  TOXINS 

The  cells  of  the  connective  tissue  in  which  the  tetanus  bacillus 
is  living  show  little  reaction,  but  the  motor  cells  of  the  central 
nervous  system,  having  a  greater  affinity  for  it,  are  profoundly 
impressed,  so  that  convulsions  of  the  controlled  muscular  system 
are  brought  about.  This  special  excitation  of  the  nerve  cells  is 
specific  because  no  other  bacterio-toxin  is  known  to  produce  it  and 
it  is  attributed  to  special  selective  affinities  of  the  nerve  cells  for 
the  poison.  This  affinity  has  its  analogue  among  the  poisons  of 
higher  plants,  thus,  strychnin  has  a  similar  selective  affinity  and  is 
also  said  to  be  specific  in  action  upon  the  motor  cells. 

The  venoms  of  various  serpents,  especially  the  cobra,  also  have 
specific  reactions,  the  cells  of  the  respiratory  centers  seeming  to 
be  most  profoundly  affected  by  them. 

The  diphtheria  bacillus,  when  observed  in  ordinary  throat  in- 
fections, is  seen  to  produce  a  pseudomembranous  angina  which 
results  in  part  from  an  irritative  local  action  of  the  organism,  which 
it  shares  in  common  with  many  others,  and  in  part  from  some 
coagulating  product  which  it  shares  in  common  with  a  few — • 
pneumococcus,  streptococcus,  etc.  Neither  of  these  reactions  is 
specific,  but  subsequent  to  these  early  manifestations  comes  de- 
pressant action  on  the  nervous  cells  with  palsy,  peculiar  to  the  prod- 
ucts of  the  diphtheria  bacillus,  and  therefore  specific. 

It  is  upon  the  peculiar  specific  reactions  of  the  bacterio-toxins 
and  the  peculiar  susceptibility  of  certain  cells  to  this  action  that  the 
production  of  distinct  clinical  manifestations  depend. 

THE  INVASION  OF  THE  BODY  BY  MICRO-ORGANISMS 

Some  iacteria  whose  invasiveness  is  insufficient  to  enable  them 
successfully  to  maintain  life  in  healthy  tissues,  occasionally  get  a 


The  Cardinal  Conditions  of  Infection  79 

foothold  in  diseased  tissues  and  assist  in  morbid  changes.  This 
is  seen  in  what  is  described  as  sapremia,  in  which  various  sapro- 
phytic  bacteria,  possessing  no  invasive  powers,  by  growing  in  the 
putrefying  tissues  of  a  gangrenous  part,  give  rise  to  poisonous  sub- 
stances which  when  absorbed  by  the  adjacent  healthy  tissues 
produce  such  constitutional  disturbances  as  depression,  fever,  and 
the  like. 

Bacteria  with  limited  invasive  powers  and  intracellular  toxins 
can  at  best  occasion  local  effects.  Such  organisms  not  infrequently 
vary,  however,  and  when  of  unusual  vitality  may  survive  entrance 
into  the  blood  and  lymph  circulations  and  occasion  bacteremia,  or, 
as  it  is  more  frequently  called,  septicemia,  a  morbid  condition 
characterized  by  the  presence  of  bacteria  in  the  circulating  blood. 
When  bacteria  entering  the  circulation  are  unable  to  pervade  the 
entire  organisms,  they  may  collect  in  the  capillaries  of  the  less  re- 
sisting tissues,  producing  local  metastatic  lesions,  usually  purulent 
in  character.  This  results  in  what  is  surgically  known  as  pyemia. 

The  mode  by  which  the  entrance  of  bacteria  into  the  circulation 
is  effected  differs  in  different  cases.  Kruse*  believes  that  they  some- 
times are  passively  forced  through  the  stomata  of  the  vessels  when 
the  pressure  of  the  inflammatory  exudate  is  greater  than  that  of 
the  blood  within  them;  that  they  may  sometimes  enter  in  to  the  bodies 
of  leukocytes  that  have  incorporated  them;  that  they  may  actually 
grow  through  the  capillary  walls,  or  that  they  reach  the  blood  cir- 
culation indirectly  by  first  following  the  course  of  the  lymphatics. 

Toxemia  results  from  the  absorption  of  the  poisonous  bacterial 
products  from  non-invasive  bacteria,  as  in  tetanus. 

THE  CARDINAL  CONDITIONS  OF  INFECTION 

Infection  can  take  place  only  when  the  micro-organisms  are 
sufficiently  virulent,  when  they  enter  in  sufficient  number,  when 
they  enter  by  appropriate  avenues,  and  when  the  host  is  susceptible 
to  their  action. 

Virulence. — Virulence  may  be  defined  as  the  disease-producing 
power  of  micro-organisms.  It  is  a  variable  quality,  and  depends 
upon  the  invasiveness  of  the  micro-organisms,  or  the  toxicity  of  their 
products,  or  both. 

A  few  bacteria  are  almost  constant  in  virulence  and  can  be  kept 
under  artificial  conditions  for  years  with  very  little  change.  Other 
bacteria  begin  to  diminish  in  virulence  so  soon  as  they  are  introduced 
to  the  artificial  conditions  of  life  in  the  test-tube.  Still  others,  and 
perhaps  the  greater  number,  can  be  modified,  and  their  virulence 
increased  or  diminished  according  to  the  experimental  manipulations 
to  which  they  are  subjected. 

Variation  in  virulence  is  not  always  a  peculiarity  of  the  species, 
*Flugge,  "Die  Mikroorganismen,"  vol.  I,  p.  271. 


8o  Infection 

for  the  greatest  differences  may  be  observed  among  individuals  of 
the  same  kind.  Thus,  the  streptococcus  usually  attenuates  rapidly 
when  kept  in  artificial  media,  so  that  special  precautions  have  to  be 
taken  to  maintain  it,  but  Hoist  observed  a  culture  whose  virulence 
was  unaltered  after  eight  years  of  continuous  cultivation  in  the 
laboratory  without  any  particular  attention  having  been  devoted 
to  it.  What  is  true  of  different  cultures  of  the  same  organisms,  is 
equally  true  of  the  individuals  in  the  same  culture.  To  determine 
such  individual  differences  is  quite  easy  among  chromogenic 
bacteria.  If  these  are  plated  in  the  ordinary  way  it  will  be  found  that 
some  colonies  are  paler  and  some  darker  than  others.  Conn  found 
that  by  repeating  the  plating  a  number  of  times  and  always  selecting 
the  palest  and  darkest  colonies  he  was  eventually  able  to  produce  two 
cultures,  one  brilliant  yellow,  the  other  colorless,  from  the  same 
original  stock  of  yellow  cocci  from  milk. 

Decrease  of  virulence  under  artificial  conditions  probably  depends 
upon  artificial  selection  of  the  organisms  in  transplantation  from 
culture  to  culture.  When  planted  upon  artificial  media,  the  vege- 
tative members  of  the  bacterial  family  proceed  to  grow  actively 
and  soon  exceed  in  number  their  more  pathogenic  fellows.  Each 
time  the  culture  is  transplanted,  more  of  the  vegetative  and  fewer 
of  the  pathogenic  forms  are  carried  over,  until  after  the  organism 
is  accustomed  to  its  new  environment,  and  grows  readily  upon  the 
artificial  media,  it  is  found  that  the  pathogenic  organisms  have 
been  largely  or  entirely  eliminated  and  the  vegetative  forms  alone 
retained. 

Increase  ot  virulence  can  be  achieved  by  artificial  selection  so 
planned  as  to  preserve  the  more  virulent  or  pathogenic  organisms 
at  the  same  time  that  the  less  virulent  and  more  vegetative  organisms 
are  eliminated.  In  cases  in  which  no  virulence  remains,  the  experi- 
mental manipulation  of  the  culture  is  directed  toward  gradual  im- 
munization of  the  micro-organisms  to  the  defensive  mechanisms  of 
the  body  of  the  animal  for  which  the  organism  is  to  be  made  virulent. 
A  number  of  methods  are  made  use  of  for  this  purpose. 

Passage  Through  Animals. — Except  in  cases  where  the  virulence 
of  the  micro-orgainsm  is  invariable,  it  is  usually  observed  that  the 
transplantation  of  the  organism  from  animal  to  animal  without 
intermediate  culture  in  vitro  greatly  augments  its  pathogenic  power. 
Of  course,  this  artificially  selects  those  members  of  the  bacterial 
family  best  qualified  for  development  in  the  animal  body,  eliminating 
the  others,  and  the  virulence  correspondingly  increases. 

The  increase  in  virulence  thus  brought  about  is,  however,  not  so 
much  an  increase  in  the  general  pathogenic  power  of  the  organism 
for  all  animals,  as  toward  the  particular  animal  or  kind  of  animal  used 
in  the  experiments.  Thus,  in  general,  the  passage  of  bacteria 
through,  mice  increases  their  virulence  for  mice,  but  not  necessarily 


The  Cardinal  Conditions  of  Infection  81 

for  cats  or  horses;  passage  through  rabbits,  the  virulence  for  rabbits, 
but  not  necessarily  for  dogs  or  pigeons,  etc. 

This  specific  character  of  the  virulence  can  be  explained  by  the 
"lateral-chain  theory  of  immunity,"  where  it  will  again  be  con- 
sidered. 

The  Use  of  Collodion  Sacs. — When  cultures  of  bacteria  are  en- 
closed in  collodion  sacs  and  placed  in  the  abdominal  or  other  body 
cavities  of  animals,  and  kept  in  this  manner  through  successive 
generations,  the  virulence  is  usually  considerably  increased.  This 
is  one  of  the  favorite  methods  used  by  the  French  investigators. 
It  keeps  the  bacteria  in  constant  contact  with  the  slightly  modified 
body  juices  of  the  animal,  which  transfuse  through  the  collodion, 
and  thus  impedes  the  development  of  such  organisms  as  are  not  able 
to  endure  their  injurious  influences.  Thus  it  becomes  only  another 
way  of  carrying  on  an  artificial  selection  of  those  members  of  the 
bacterial  family  that  can  endure,  and  eliminating  those  that  cannot 
endure  the  defensive  agencies  of  those  juices  with  which  the  organ- 
isms come  in  contact.* 

The  addition  of  animal  fluids  to  the  culture-media  sometimes 
enables  the  investigator  to  increase,  and  usually  enables  him  to 
maintain,  the  virulence  of  bacteria.  A  series  of  generations  in 
gradually  increasing  concentrations  of  the  body  fluid  should  be 
employed,  until  the  organism  becomes  thoroughly  accustomed  to  it. 

In  some  cases  it  may  be  sufficient  to  use  a  single  standard  mixture, 
thus:  Shawf  found  that  he  could  exalt  the  virulence  of  anthrax 
bacilli  by  cultivating  them  upon  blood-serum  agar  for  fourteen 
generations,  after  which  they  were  three  times  as  active  as  cultures 
similarly  transferred  upon  ordinary  agar-agar. 

The  increase  of  virulence  under  such  conditions  probably  depends 
upon  the  immunization  of  the  bacteria  to  the  body  juices  of  the 
animals,  and  this  whole  matter  will  be  understood  after  the  subject 
" Immunity"  has  been  considered. 

Number. — The  number  of  bacteria  entering  the  infected  animal 
has  a  very  important  bearing  upon  infection. 

The  entrance  of  a  single  micro-organism  of  any  kind  is  scarcely 
ever  able  to  cause  infection  because  of  the  uncertainty  of  its  being 
able  to  withstand  the  changed  conditions  to  which  it  is  subjected. 
In  most  cases  a  considerable  number  of  organisms  is  necessary  in 
order  that  some  may  survive.  Park  points  out  that  when  bacteria 
are  transplanted  from  culture  to  culture,  under  conditions  supposed 
to  be  favorable,  many  of  them  die.  It  seems  not  improbable,  there- 
fore, that  when  they  are  transplanted  to  an  environment  in  which  are 
present  certain  mechanisms  for  defending  the  organism  against  them, 
many  more  must  inevitably  die.  The  more  virulent  an  organism 
is,  the  fewer  will  be  the  number  required  to  infect.  Marmorek, 

*  Directions  for  making  and  using  the  capsules  are  given  in  the  chapter  upon 
Animal  Experimentation. 

f  "Brit.  Med.  Jour.,"  May  9,  1903. 
6 


82  Infection 

in  his  experiments  with  antistreptococcic  serum,  used  a  streptococcus 
whose  virulence  was  exalted  by  passage  through  rabbits  and  in- 
termediate cultivation  upon  agar-agar  containing  ascitic  fluid,  until 
one  hundred  thousand  millionth  of  a  cubic  centimeter  (un  cent 
milliardieme)  was  fatal  for  a  rabbit.  In  this  quantity  it  is  scarcely 
probable  that  more  than  a  single  coccus  could  have  been  present. 
Single  anthrax  or  glanders  bacilli  may  infect  rabbits  and  guinea- 
pigs.  Roger  found  that  820  tubercle  bacilli  from  the  culture  with 
which  he  experimented  were  required  to  infect  a  guinea-pig,  when 
introduced  beneath  the  skin.  Herman  found  that  it  required  4  or 
5  cc.  of  a  culture  of  Staphylococcus  pyogenes  to  produce  suppura- 
tion in  the  peritoneal  cavity  of  an  animal;  0.75  cc.  to  produce  it 
beneath  the  skin;  0.25  cc.  in  the  pleura;  0.05  cc.  in  the  veins  and 
o.oooi  cc.  in  the  anterior  chamber  of  the  eye. 

In  experimenting  with  Bacillus  proteus  vulgaris,  Watson  Cheyne 
found  that  5,000,000  to  6,000,000  organisms  injected  beneath  the 
skin  did  not  produce  any  lesion;  8,000,000  caused  the  formation 
of  an  abscess;  56,000,000  produced  a  phlegmon  from  which  the 
animal  died  in  five  or  six  weeks  and  225,000,000  were  required  to 
cause  the  death  of  the  animal  in  twenty-four  hours.  In  studying 
Staphylococcus  aureus  upon  rabbits  he  found  that  25,000,000 
would  cause  an  abscess,  but  1,000,000,000  were  necessary  to  cause 
death. 

The  Avenue  of  Infection. — The  successful  invasion  of  the  body 
by  certain  bacteria  can  be  achieved  only  when  they  enter  it  through 
appropriate  avenues.  Even  when  invasion  is  possible  through 
several  channels,  the  parasite  most  commonly  invades  through  one 
that  may,  therefore,  be  regarded  as  most  appropriate,  and  furnishes 
the  typical  picture  of  the  infection. 

Thus,  gonococci  usually  reach  the  body  through  the  urogenital 
mucous  membranes,  where  they  set  up  the  various  inflammatory 
reactions  collectively  known  as  gonorrhea — i.e.,  urethritis,  vaginitis, 
prostatitis,  orchitis,  cystitis,  etc.  These  constitute  the  typical 
picture  of  the  infection.  The  organism  may  also  successfully  invade 
the  conjunctiva,  producing  blennorrhea,  but  there  is  no  evidence 
that  gonococci  can  successfully  invade  the  body  through  the  skin, 
the  respiratory,  or  alimentary  mucous  membrane. 

Typhoid  and  cholera  infections  seem  to  take  place  through  the 
alimentary  mucous  membrane,  and  the  evidence  that  infection  takes 
place  by  inhalation  is  slight.  It  is  not  known  to  take  place  through 
the  urogenital  system,  the  conjunctiva,  or  the  skin. 

The  avenue  of  entrance  not  only  determines  infection,  but  may 
also  determine  the  form  that  it  takes.  Thus,  tubercle  bacilli  rubbed 
into  the  deeper  layer  of  the  skin  produce  a  chronic  inflammatory 
disease,  called  lupus,  that  lasts  for  years  and  rarely  results  in 
generalised  tuberculosis.  Bacilli  reaching  the  cervical  or  other 
lymph-nodes  by  entrance  through  the  tonsils,  may  remain  localized, 


The  Cardinal   Conditions  of  Infection  83 

producing  enlargement  and  softening  of  the  nodes,  or  passing  through 
them  reach  the  circulation,  in  which  they  may  be  carried  to  the 
bones  and  joints  and  occasion  chronic  inflammation  with  necrosis 
and  ultimate  evacuation  or  exfoliation  of  the  diseased  mass,  after 
which  the  patient  may  recover.  Bacilli  entering  the  intestine  in 
many  cases  produce  implantation  lesions  in  the  intestinal  walls; 
bacilli  inhaled  into  the  lung,  or  conveyed  to  it  from  the  intestine 
by  the  thoracic  duct  and  veins,  produce  the  ordinary  pulmonary 
tuberculosis  known  as  phthisis  or  consumption. 

Inhaled  pneumococci  colonizing  in  the  pharynx  have  been  known 
to  produce  pseudomembranous  angina;  in  the  lungs,  pneumonia; 
implanted  upon  the  conjunctiva,  conjunctivitis.  In  these  cases  we 
can  look  upon  the  type  of  infection  as  depending  upon  the  portal 
through  which  the  invading  organism  found  its  way  into  the  tissues. 

The  avenue  of  entrance  is,  for  obvious  reasons,  less  important 
when  the  micro-organism  is  of  some  rapidly  invasive  form,  whose 
chief  operation  is  in  the  streaming  blood  or  in  the  lymphatics. 
Anthrax  in  most  animals  is  characterized  by  a  bacteremia  regardless 
of  the  point  of  primary  infection.  Bubonic  plague  rapidly  becomes 
a  bacteremia  regardless  of  the  entrance  of  the  Bacillus  pestis  by  in- 
halation into  the  lungs,  or  by  way  of  the  lymphatics  through  super- 
ficial lesions.  The  failure  of  the  micro-organisms  to  colonize 
successfully  when  introduced  through  inappropriate  avenues  may 
be  explained  by  a  consideration  of  the  local  conditions  to  which 
they  are  subjected. 

When  they  are  introduced  beneath  the  skin,  bacteria  are,  in  most 
cases,  delayed  in  reaching  the  circulation,  and  are  in  the  meantime 
subjected  to  the  germicidal  action  of  the  lymph  and  exposed  to  the 
attacks  of  phagocytes.  Many  succumb  to  these  and  never  penetrate 
more  deeply  into  the  body.  Should  any  survive,  they  may  be  trans- 
ported to  the  lymph-nodes  and  there  destroyed,  or,  passing  through 
these  barriers  without  destruction,  and  reaching  the  venous  channels, 
they  have  next  to  pass  through  the  pulmonary  capillaries,  where 
they  are  apt  to  be  caught  and  destroyed.  Finally,  should  any  es- 
cape all  these  defenses  and  reach  the  general  circulation,  it  is  to  find 
the  endothelium  of  the  capillaries  prone  to  collect  and  detain  them 
until  destruction  is  finally  effected.  The -systemic  circulation  is 
also  defended  against  such  micro-organisms  as  might  reach  the  veins 
through  lesions  or  accidents  of  the  abdominal  viscera,  by  the  inter- 
position of  the  portal  capillary  network  of  the  liver,  where  the  bac- 
teria are  caught  and  many  of  them  destroyed,  or  passing  which,  the 
pulmonary  capillary  system  acts  as  a  second  barrier  against  them. 
The  deeper  the  penetration,  the  more  active  the  defense  becomes, 
the  blood  itself  furnishing  agglutinins,  bacterio-lysins,  and  phago- 
cytes for  the  destruction  of  the  micro-organisms  and  the  protection 
of  the  host. 

These  defenses,  however,  are  of  no  avail  against  actively  invasive 


84  Infection 

organisms  provided  with  the  means  of  overcoming  them  all  through 
aggressins  that  destroy  the  germicidal  humors  or  toxins  that  kill 
or  paralyze  the  cells.  When  these  are  injected  directly  into  the 
streaming  blood  they  produce  their  effects  more  rapidly  than  when 
injected  beneath  the  skin  or  elsewhere,  because  the  field  of  operation 
is  immediately  reached  instead  of  through  a  roundabout  course  in 
which  so  many  defenses  have  to  be  overcome.  Taking  anthrax 
bacilli,  whose  invasiveness  has  already  been  dwelt  upon,  as  an 
example,  Roger*  found  that  when  the  orginisms  were  injected  into 
the  aorta,  animals  died  more  quickly  than  when  they  were  injected 
into  the  veins  and  obliged  to  find  their  way  through  the  pulmonary 
capillaries  to  the  general  circulation.  If  the  injections  were  made 
into  the  portal  vein,  the  animals  stood  a  good  chance  of  recovery, 
the  liver  possessing  the  power  of  destroying  sixty-four  times  as  many 
anthrax  bacilli  as  would  prove  fatal  if  introduced  through  other 
channels. 

The  conditions  differ,  however,  in  different  infections,  for  when 
Roger  experimented  with  streptococci  instead  of  anthrax  bacilli, 
he  found  that  if  the  bacilli  were  inoculated  into  the  portal  vein  the 
animals  died  more  quickly  than  when  they  were  injected  into  the 
aorta,  and  that  when  the  bacilli  were  injected  into  the  peripheral 
veins  the  animals  lived  longest,  the  liver  seeming  to  be  far  less 
destructive  to  streptococci  than  the  lungs. 

The  Susceptibility  of  the  Host. — Susceptibility  is  liability  to  in- 
fection. It  is  a  condition  in  which  the  host  is  unable  to  defend  itself 
against  invading  micro-organisms.  Unusual  or  unnatural  suscep- 
tibility is  also  spoken  of  as  predisposition  or  dyscrasia. 

Many  animals  and  plants  are  naturally  without  any  means  of 
overcoming  the  invasiveness  of  certain  parasitic  micro-organisms, 
and  are,  therefore,  naturally  susceptible;  others  naturally  resist 
their  inroads,  but  through  various  temporary  or  permanent  physio- 
logic changes  may  lose  the  defensive  power. 

In  general,  it  is  true  that  any  condition  that  depresses  or  diminishes 
the  general  physiological  activity  of  an  animal  diminishes  its  ability 
to  defend  itself  against  the  pathogenic  action  of  bacteria,  and  so 
predisposes  to  infection.  These  changes  are  often  so  subtile  that 
they  escape  detection,  though  at  times  they  can  be  partly  understood. 

The  inhalation  of  noxious  vapors.  It  has  long  been  supposed 
that  sewer  gas  was  responsible  for  the  occurrence  of  certain  in- 
fectious diseases,  and  when  the  nature  of  these  diseases  was  made 
clear  by  a  knowledge  of  their  bacterial  causes,  the  old  belief  still 
remained  and  many  sanitarians  continued  to  believe  that  defective 
sewage  is  in  some  way  connected  with  their  occurrence.  It  is 
difficult  to  prove  or  disprove  the  matter  experimentally.  Men  who 
work  in  sewers  and  plumbers  who  breathe  much  sewer  gas  are 
not  apparently  affected  by  it.  Alessif  found  that  rats,  rabbits,  and 

*  "Introduction  to  the  Study  of  Medicine,"  p.  151. 
f  "Centralbl.  f.  Bakt.,"  etc.,  1894,  xv,  p.  228. 


The  Cardinal  Conditions  of  Infection  85 

guinea-pigs  kept  in  cages  some  of  which  were  placed  over  the  open- 
ing of  a  privy,  while  in  others  the  excreta  of  the  animals  were 
allowed  to  accumulate,  suffered  from  a  pronounced  diminution  of 
the  resisting  powers.  This  would  seem  to  be  inconsistent  with  the 
habits  of  rats,  many  of  which  live  in  sewers.  Abbott*  caused  rabbits 
to  breathe  air  forced  through  sewage  and  putrid  meat  infusions 
for  one  hundred  and  twenty-nine  days,  and  found  that  the  products 
of  decomposition  inhaled  by  the  animals  played  no  part  in  producing 
disease,  or  in  inducing  susceptibility  to  it. 

Fatigue  is  a  well-recognized  clinical  cause  of  susceptibility  to 
disease,  and  experimental  evidence  of  its  correctness  is  not  wanting. 
Charrin  and  Rogerf  found  that  white  rats,  which  naturally  resist 
infection  with  anthrax,  succumbed  to  the  infection  if  compelled  to 
turn  a  revolving  wheel  until  exhausted  before  inoculation. 

Exposure  to  cold  seriously  diminishes  the  resisting  power  of  the 
warm-blooded  animals.  It  is  an  everyday  experience  that  chilling 
the  body  predisposes  to  "cold"  and  may  be  the  starting-point  of 
pneumonia.  Pasteur  found  that  fowls,  which  resist  anthrax  under 
normal  conditions,  succumbed  to  infection  if  kept,  for  some  time,  in 
a  cold  bath  before  inoculation. 

The  reverse  seems  to  be  true  of  the  cold-blooded  animals,  for 
Gibier  J  found  that  frogs,  naturally  resistant  to  the  anthrax  bacillus, 
would  succumb  to  infection  if  kept  at  37°C.  after  inoculation. 

Diet  produces  some  variation  in  the  resisting  powers.  The 
tendency  of  scorbutics  to  suffer  from  infectious  disorders  of  the  mouth, 
the  frequency  with  which  epidemics  of  infectious  disease  follow 
famines,  and  the  enterocolitis  of  marasmatic  infants,  illustrate  the 
effects  of  insufficient  food  in  predisposing  to  disease.  We  also  find 
that  the  infectious  diseases  of  carnivorous  animals  are  not  the  same 
as  those  of  herbivorous  animals,  and  that  the  former  are  exempt 
from  many  disorders  to  which  the  latter  quickly  succumb.  Hankin 
was  able  to  show  experimentally  that  meat-fed  rats  resisted  anthrax 
infection  far  better  than  rats  fed  upon  bread. 

Intoxication  of  all  kinds  predisposes  to  infection.  Platania§ 
found  that  such  animals  as  frogs,  pigeons,  and  dogs  became  sus- 
ceptible to  anthrax  when  under  the  influence  of  curare,  chloral, 
and  alcohol.  Leo||  found  that  white  rats  fed  upon  phloridzin  became 
susceptible  to  anthrax.  Wagner**  found  that  pigeons  become  sus- 
ceptible to  anthrax  when  under  the  influence  of  chloral.  Abbottff 
found  the  resisting  powers  of  rabbits  against  Streptococcus  pyogenes 
and  Bacillus  coli  diminished  by  daily  intoxication  with  5  to  15  c.c. 

*  "Trans.  Assoc.  Amer.  Phys.,"  1895. 
f  "Compte  rendu  Soc.  de  Biol  de  Paris,"  Jan.  24,  1890. 
j  "Compte  rendu  Acad.  des  Sciences  de  Paris,"  1882,  t.  xcix,  p.  1605. 
§  See  Sternberg's  "Immunity  and  Serum  Therapy,"  p.   10;  "Centralbl.   f. 
Bakt.,"  etc.,  Bd.  vn,  p.  405. 

||  "Zeitschrift  fur  Hyg.,"  1889,  Bd.  vn,  p.  505. 

**"Wratsch,"  1890,  39,  40. 

ft  "Jour,  of  Exp.  Med.,"  1896,  vol.  i,  No.  3. 


86  Infection 

of  alcohol  introduced  into  the  stomach  through  a  tube.  Salant* 
found  that  alcohol  was  disadvantageous  in  combating  the  infectious 
diseases  because  it  diminished  the  glycogen  content  of  the  liver  which 
Collaf  had  found  an  important  adjunct  in  supporting  the  resisting 
power. 

It  is  a  common  clinical  observation  that  excessive  indulgence 
in  alcohol  predisposes  to  certain  infections,  notably  pneumonia, 
and  every  surgeon  knows  the  danger  of  pneumonia  after  anesthetiza- 
tion with  ether. 

Traumatic  injury  and  mutilation  of  the  body  are  not  without 
effect  upon  infection.  The  more  extensive  the  damage  done  to 
the  tissues,  the  greater  the  danger  of  infection,  and  the  more  serious 
the  consequences  of  infection  when  it  takes  place. 

The  mutilation  of  the  body  by  the  removal  of  certain  organs  is 
of  disputed  importance.  There  is  much  literature  upon  the  effect 
of  the  spleen  in  overcoming  infectious  agents,  but  the  experimental 
evidence  seems  about  equally  divided  as  to  whether  an  animal  is 
more  or  less  susceptible  after  the  removal  of  this  organ  than  it  was 
before. 

Morbid  conditions  in  general  predispose  to  infection.  The  fre- 
quency with  which  diabetics  suffer  from  furuncles,  carbuncles,  and 
local  gangrenous  lesions  of  the  skin;  the  increased  susceptibility  of 
phthisics  to  bronchopneumonia  of  other  than  tuberculous  origin; 
the  apparent  predisposition  of  injured  joints  and  pneumonic  lungs 
to  tuberculosis;  the  extensive  streptococcus  invasions  accompany- 
ing scarlatina  and  variola;  the  presence  of  Bacillus  icteroides  and 
various  other  organisms  in  the  blood  and  tissues  of  yellow  fever 
patients,  and  the  presence  of  Bacillus  suipestifer  in  the  bodies  of 
hogs  suffering  with  hog  cholera,  all  show  the  diminution  in  the  gen- 
eral resisting  power  of  an  individual  already  diseased. 

MIXED  INFECTIONS 

The  general  prevalence  of  bacteria  determines  that  few  can 
enter  and  infect  the  body  of  a  host  without  the  association  of  other 
kinds.  Therefore  their  operation  in  the  body  is  subject  to  modifica- 
tions produced  in  them  or  in  the  host  by  these  associated  organisms. 

In  experimental  investigations  this  fact  is  not  infrequently  for- 
gotten, and  it  is  often  remarked  with  surprise  that  the  results  of 
inoculation  with  pure  cultures  of  a  micro-organism  may  be  clinically 
different  from  those  observed  under  natural  conditions. 

The  tetanus  bacillus,  which  endures  with  difficulty  the  effects 
of  uncombined  oxygen,  flourishes  in  association  with  saprophytic 
organisms  by  which  the  oxygen  is  absorbed.  The  same  thing  is 
probably  true  of  other  obligatory  anaerobic  organisms. 

*  "Jour.  Amer.  Med.  Assoc.,"  1906,  XLVII,  18,  Nov.  3,  p.  1467. 
f  "Archiv.  Ital.  de  Biologic,"  xxvi. 


Mixed  Infections  87 

The  metabolic  products  of  one  species  may  intensify  or  accelerate 
the  action  of  those  of  an  associated  species,  or  the  reverse  may  be 
true,  and  the  products  of  different  organisms,  having  different 
chemical  composition,  may  neutralize  one  another,  or  combine  to 
form  some  entirely  new  substance  which  is  entirely  different  from 
its  antecedents.  Such  conditions  cannot  fail  to  influence  the  type 
and  course  of  infection. 


CHAPTER  IV 

IMMUNITY 

IMMUNITY  is  ability  to  resist  infection.  It  is  the  ability  of  an 
organism  successfully  to  antagonize  the  invasive  powers  of  parasites, 
or  to  annul  the  injurious  properties  of  their  products.  The  mech- 
anism of  immunity  is  complicated  or  otherwise  according  to  cir- 
cumstances. When  the  invasive  action  of  non-toxicogenic  bacteria 
is  to  be  overcome,  certain  reactions,  mostly  on  the  part  of  the  phago- 
cytic  cells,  are  called  into  action;  when  the  toxic  products  of  bacteria 
are  to  be  deprived  of  injurious  effects,  the  reaction  seems  to  take 
place  between  the  toxin  and  certain  combining  and  neutralizing 
substances  contained  in  the  body  juices;  when  bacterial  invasion 
and  intoxication  are  both  to  be  antagonized,  both  mechanisms  are 
engaged  in  the  defenses,  comparatively  simple  or  exceedingly  com- 
plex, according  to  the  conditions  involved.  The  more  involved 
the  conditions  of  infection  become,  the  more  complicated  the 
defensive  reactions  become,  until  it  may  no  longer  be  possible 
accurately  to  analyze  them. 

Some  have  endeavored  to  refer  all  of  the  phenomena  of  im- 
munity to  the  ability  of  the  animal  to  endure  the  bacterio-toxins, 
and  have  sought  to  relegate  the  reactions  against  invasion  to  a 
subsidiary  place.  This  is  undoubtedly  an  error,  as  the  mechanisms 
are  different  and  the  prompt  action  of  one  may  make  the  action  of 
the  other  unnecessary.  Metschnikoff*  found  that  frogs  injected 
with  0.5  cc.  of  cholera  toxin  died  promptly,  but  that  frogs  injected 
with  cultures  of  the  cholera  spirillum  recovered  without  illness. 
This  would  suggest  that  the  recovery  of  the  infected  frog  depended 
upon  some  defensive  mechanism  combating  the  invasiveness  of  the 
bacteria  and  so  preventing  the  production  of  the  toxin  to  which  the 
frog  was  susceptible. 

Immunity  must  not  be  conceived  as  something  inseparably 
associated  with  infection.  The  reactions  of  the  body  toward 
bacteria  in  the  infectious  diseases  are  identical  with  those  toward 
other  minute  irritative  bodies,  and  the  reactions  toward  bacterio- 
toxins  are  identical  with  those  toward  other  toxic  substances,  so 
that  the  only  way  by  which  a  satisfactory  understanding  of  the 
phenomena  can  be  reached  is  by  carefully  comparing  the  reactions 
produced  by  bacteria  and  their  products  with  those  produced  by 
other  active  bodies. 

*  "Immunite  dans  les  Maladies  Infectieuses,"  Paris,  1901,  p.  150. 

88 


Natural  Immunity  89 

Immunity  is  called  active  when  the  animal  protects  itself  through 
its  own  activities,  passive  when  the  protection  depends  upon  defen- 
sive substances  prepared  by  some  other  animal  entering  into  it. 
Thus,  if  a  frog  be  injected  with  anthrax  bacilli,  its  leukocytes  de- 
vour the  bacteria,  destroy  them,  and  so  protect  the  frog  from  in- 
fection; the  immunity  is  active  because  it  depends  upon  the  activity 
of  the  frog's  phagocytes.  But  if  a  guinea-pig  previously  given  anti- 
tetanic  serum  be  injected  with  tetanus  toxin,  and  so  recovers  from 
the  toxin,  the  resisting  power,  conferred  by  the  antitoxin  previously 
injected,  does  not  depend  upon  any  activity  of  the  animal,  which 
remains  entirely  passive. 

Immunity  is  largely  relative.  Fowls  are  immune  against  tetanus, 
that  is,  they  can  endure,  without  injury,  as  much  toxin  as  tetanus 
bacilli  can  produce  in  their  bodies,  and  suffer  no  ill  effects  from  in- 
oculation. If,  however,  a  large  quantity  of  tetanotoxin  produced 
in  a  test-tube  be  introduced  into  their  bodies,  they  succumb  to  it. 
Mongooses  and  hedgehogs  are  sufficiently  immune  against  the 
venoms  of  serpents  to  resist  as  much  poison  as  is  ordinarily  injected 
by  the  serpents,  but  by  collecting  the  venom  from  several  serpents 
and  injecting  considerable  quantities  of  it,  both  animals  can  be  killed. 
Rats  cannot  be  killed  by  infection  with  Bacillus  diphtherias,  and 
Cobbett*  found  that  they  could  endure  from  1500  to  1800  times  as 
much  diphtheria  toxin  as  guinea-pigs,  though  more  than  this  would 
kill  them. 

Carl  Frankel  has  expressed  the  whole  matter  very  forcibly  when 
he  says :  "A  white  rat  is  immune  against  anthrax  in  doses  sufficiently 
large  to  kill  a  rabbit,  but  not  necessarily  against  a  dose  sufficiently 
large  to  kill  an  elephant." 

NATURAL  IMMUNITY 

Natural  immunity  is  the  natural,  inherited  resistance  against 
infection  or  intoxication,  peculiar  to  certain  groups  of  animals,  and 
common  to  all  the  individuals  of  those  groups. 

Few  micro-organisms  are  capable  of  infecting  all  kinds  of  animals; 
indeed,  it  is  doubtful  whether  any  known  organism  possesses  such 
universally  invasive  powers. 

The  micro-organisms  of  suppuration  seem  able  to  infect  animals 
of  many  different  kinds,  sometimes  producing  local  lesions,  some- 
times invading  rapidly  with  resulting  bacteremia.  The  tubercle 
bacillus  is  known  to  be  pathogenic  for  mammals,  birds,  reptiles, 
batrachians,  and  fishes,  though  it  is  still  uncertain  whether  the 
infecting  organisms  in  these  cases  are  identical  or  slightly  differing 
species. 

As  a  rule,  however,  the  infectivity  of  bacteria  and  other  micro- 
organisms is  restricted  to  certain  groups  of  animals  which  usually 
*"Brit.  Med.  Jour.,"  April  15,  1899. 


90  Immunity 

have  more  or  less  resemblance  to  one  another;  thus,  anthrax  is 
essentially  a  disease  of  warm-blooded  animals,  though  certain 
exceptions  are  observed,  and  Metschnikoff  has  found  that  hippo- 
campi (sea-horses),  perch,  crickets,  and  certain  mussels  are  sus- 
ceptible. Among  the  warm-blooded  animals  anthrax  is  most  fre- 
quent among  the  herbivora,  though  some  carnivora  may  also  be 
infected. 

Close  relationship  is  not,  however,  a  guarantee  that  animals 
will  behave  similarly  toward  infection.  The  rabbit,  guinea-pig, 
and  the  rat  are  rodents,  but  though  the  rabbit  and  guinea-pig  are 
susceptible  to  anthrax,  the  rat  is  immune.  This  is  still  better 
exemplified  in  the  susceptibility  of  mice  to  glanders.  The  field- 
mouse  seems  to  be  the  most  susceptible  of  all  animals  to  infection 
with  Bacillus  mallei;  the  house  mouse  is  much  less  susceptible,  and 
the  white  mouse  is  immune.  Mosquitos,  though  closely  related, 
are  different  in  their  immunity  to  the  malarial  parasite.  The 
culex  does  not  harbor  the  parasite  at  all,  and  of  the  anopheles,  two 
very  similar  species  seem  to  behave  very  differently.  Anopheles 
maculipennis  being  the  common  definitive  host  of  the  parasite, 
while  Anopheles  punctipennis  is  not  known  to  be  susceptible  to  it. 
The  same  differences  may  exist  among  the  members  of  the  human 
species.  It  has  been  asserted  that  Mongolians,  and  especially 
Japanese,  are  immune  against  scarlatina,  and  that  negroes  are 
immune  against  yellow  fever,  but  increasing  information  is  to  the 
contrary. 

Human  beings  suffer  from  typhoid,  cholera,  measles,  scarlatina, 
yellow  fever,  varicella,  and  numerous  other  diseases  unknown  among 
the  lower  animals,  even  those  domestic  animals  with  which  they 
come  in  close  contact.  They  also  suffer  from  Malta  fever,  anthrax, 
rabies,  glanders,  bubonic  plague,  and  tuberculosis,  which  are  common 
among  the  lower  animals.  Animals,  in  turn,  suffer  from  distemper, 
septicemia,  etc.,  the  respective  micro-organisms  of  which  are  not 
known  to  infect  man. 

It  has  already  been  pointed  out  that  mongooses  and  hedgehogs 
are  immune  against  the  venom  of  serpents  from  which  other  animals 
quickly  die.  The  tobacco-worm  lives  solely  upon  tobacco-leaves, 
the  juice  of  which  is  intensely  poisonous  to  higher  animals,  and  is 
also  a  good  insecticide.  Boxed  cigars  and  baled  tobacco  are  often 
ruined  by  the  larvae  of  a  small  beetle  that  feeds  upon  them,  and  a 
glance  over  the  poisonous  vegetables  will  show  that  few  of  them 
escape  the  attacks  of  insects  immune  against  their  juices. 

These  facts  are  sufficient  to  show  that  many  animals  are  by 
nature  immune  against  the  invasion  of  microparasites  of  certain 
kinds,  and  that  they  are  also  at  times  immune  against  poisons. 
Immunity  against  one  kind  of  infection  or  intoxication  is,  however, 
entirely  independent  of  all  other  infections  and  intoxications.  Im- 
munity against  infection  usually  guarantees  exemption  from  the 


Acquired  Immunity  91 

toxic  products  of  that  particular  micro-organism,  though  experi- 
ment may  show  the  animal  to  be  susceptible  to  it.  Immunity 
against  any  form  of  bacterio-toxin  usually,  though  not  necessarily, 
determines  that  the  micro-organism,  though  it  may  be  able  to  invade 
the  body,  can  do  very  little  harm. 

ACQUIRED  IMMUNITY 

Acquired  immunity  is  resistance  against  infection  or  intoxica- 
tion possessed  by  certain  animals,  of  a  naturally  susceptible  kind, 
in  consequence  of  conditions  peculiar  to  them  as  individuals.  It  is  a 
peculiarity  of  the  individual,  not  of  his  kind,  and  signifies  a  subtile 
change  in  physiology  by  which  latent  defensive  powers  are  stimulated 
to  action.  The  reactions  in  general  correspond  with  those  of  natural 
immunity,  and  comprise  mechanisms  for  overcoming  the  invasion 
of  pathogenic  organisms,  for  neutralizing  or  destroying  their  toxins 
or  for  both.  As  an  acquired  character  and  an  individual  peculiarity 
it  is  not  transmitted  to  the  offspring,  though  these  sometimes  also 
acquire  immunity  through  the  parents.  Thus  in  studying  im- 
munity of  mice  against  ricin,  Ehrlich  found  that  the  newly  born 
offspring  of  an  immune  mother  were  not  immune,  though  they 
subsequently  became  so  through  her  milk. 

Acquired  immunity  differs  from  natural  immunity  in  being  more 
variable  in  degree  and  duration.  The  animal  may  be  immune 
to-day,  but  lose  all  power  of  defending  itself  a  month  hence. 

Natural  immunity  is  always  active,  but  certain  forms  of  acquired 
immunity  are  passive. 

Immunity  may  be  acquired  through  infection  or  intoxication,  and 
in  either  case  may  be  accidental  or  experimental. 

(A)  Active  Acquired  Immunity. — i.  Immunity  Acquired  through 
Infection. — (a)  Accidental  Infection. — The  most  familiar  form  of 
acquired  immunity  follows  an  attack  of  an  infectious  disease.  Every 
one  knows  that  an  attack  of  measles,  scarlatina,  varicella,  variola, 
yellow  fever,  typhoid  fever,  and  other  common  infectious  maladies, 
is  a  fairly  good  guarantee  of  future  exemption  from  the  respective 
disease.  Immunity  thus  acquired  is  not  transmissible  to  the  off- 
spring. Almost  everybody  has  had  measles,  yet  almost  all  children 
are  born  susceptible  to  it.  It  is  not  necessarily  permanent,  as  is 
shown  by  the  not  infrequent  cases  in  which  second  attacks  of  measles 
occur.  In  some  cases,  as  after  typhoid  fever,  the  immunity  is  not 
at  first  observable  and  the  patient  may  suffer  from  relapses.  Later 
it  becomes  well-established  and  no  repetition  of  the  disease  is  possi- 
ble for  years. 

Sometimes  the  infection,  by  which  immunity  is  acquired,  is  not 
exactly  similar  to  the  disease  against  which  it  affords  protection, 
as  in  the  case  of  vaccinia,  which  protects  against  variola.  It  is 
still  controversial,  however,  whether  cow-pox  is  variola  of  the  cow 


92  Immunity 

or  an  entirely  different  disease.  Cow-pox  was,  however,  common 
in  the  days  when  smallpox  was  frequent,  and  has  now  become 
extremely  rare. 

(b)  Experimental  Infection. — I.  Inoculation:  This  is  an  attempt 
to  prevent  the  occurrence  of  a  fatal  attack  of  an  infectious  disease, 
by  inducing  a  mild  attack  of  the  same  disease  when  the  individual 
is  in  good  health,  and  at  his  maximum  resisting  power.  The  oldest 
experiments  date  from  unknown  antiquity  and  were  practised 
in  China  and  other  Oriental  countries  for  the  purpose  of  preventing 
smallpox.  The  Chinese  method  of  experimentally  producing 
variolous  infection  was  very  crude  and  consisted  in  introducing  crusts 
from  cases  of  variola  into  the  nose,  and  tying  them  upon  the  skin. 
The  Turkish  method  was  much  more  neat,  in  that  a  small  quantity 
of  the  variolous  pus  was  introduced  into  a  scarification  upon  the  skin 
of  the  individual  to  be  protected.  The  following  extract  is  from  a 
letter  of  Lady  Montague,*  wife  of  the  British  Ambassador  to 
Turkey,  who  brought  the  so-called  "  inoculation "  method  from 
Turkey  in  the  early  part  of  the  eighteenth  century  (1718): 

"  .  .  .  .  .  Apropos  of  distempers,  I  am  going  to  tell  you  a  thing  that  I  am 
sure  will  make  you  wish  yourself  here.  The  smallpox,  so  fatal,  and  so  general 
amongst  us,  is  here  entirely  harmless  by  the  invention  of  ingrafting,  which  is 
the  term  they  give  it.  There  is  a  set  of  old  women  who  make  it  their  business 
to  perform  the  operation  every  autumn,  in  the  month  of  September,  when  the 
great  heat  is  abated.  People  send  to  one  another  to  know  if  any  of  their  family 
has  a  mind  to  have  the  smallpox;  they  make  parties  for  this  purpose,  and  when 
they  are  met  (commonly  fifteen  or  sixteen  together) ,  the  old  woman  comes  with 
a  nut-shell  full  of  the  matter  of  the  best  sort  of  smallpox,  and  asks  what  vein 
you  please  to  have  opened.  She  immediately  rips  open  that  you  offer  to  her 
with  a  large  needle  (which  gives  you  no  more  pain  than  a  common  scratch),  and 
puts  into  the  vein  as  much  venom  as  can  lie  upon  the  head  of  her  needle,  and 
after  binds  up  the  little  wound  with  a  hollow  bit  of  shell;  and  in  this  manner 
opens  four  or  five  veins.  The  Grecians  have  commonly  the  superstition  of 
opening  one  in  the  middle  of  the  forehead,  in  each  arm,  and  on  the  breast,  to 
mark  the  sign  of  the  cross;  but  this  has  a  very  ill  effect,  all  these  wounds  leaving 
little  scars,  and  is  not  done  by  those  that  are  not  superstitious,  who  choose  to  have 
them  in  the  legs,  or  that  part  of  the  arm  that  is  concealed.  The  children  of  young 
patients  play  together  all  the  rest  of  the  day,  and  are  in  perfect  health  to  the 
eighth.  Then  the  fever  begins  to  seize  them,  and  they  keep  their  beds  two  days, 
very  seldom  three.  They  have  very  rarely  above  twenty  or  thirty  [pocks]  in 
their  faces,  which  never  mark;  and  in  eight  days'  time  they  are  as  well  as  before 
their  illness.  Where  they  are  wounded,  there  remain  running  sores  during  the 
distemper,  which  I  don't  doubt  is  a  great  relief  to  it.  Every  year  thousands 
undergo  this  operation;  and  the  French  embassador  says  pleasantly,  that  they 
take  the  smallpox  here  by  way  of  diversion,  as  they  take  the  waters  in  other 
countries.  There  is  no  example  of  any  one  that  has  died  in  it;  and  you  may 
believe  I  am  very  well  satisfied  of  the  safety  of  this  experiment,  since  I  intend  to 
try  it  on  my  dear  little  son. 

"I  am  patriot  enough  to  take  pains  enough  to  bring  this  useful  invention  into 
fashion  in  England;  and  I  should  not  fail  to  write  to  some  of  our  doctors  very 
particularly  about  it,  if  I  knew  any  one  of  them  that  I  thought  had  virtue  enough 
to  destroy  such  a  considerable  branch  of  their  revenue  for  the  good  of  mankind. 
But  that  distemper  is  too  beneficial  to  them  not  to  expose  to  all  their  resentment 
the  hardy  wight  that  should  undertake  to  put  an  end  to  it." 

*  See  the  "Letters  of  Lady  Mary  Wortley  Montague;"  letter  to  Miss  Sarah 
Chisives  dated  Adrianople,  April  i  (O.  S.),  1717. 


Vaccination  93 

By  both  methods  the  very  disease,  variola,  against  which  protec- 
tion was  desired,  was  induced,  the  only  advantage  of  the  experi- 
mental over  the  accidental  infection  being  that  by  selecting  the 
infective  virus  from  a  mild  case  of  variola,  by  performing  the 
operation  at  a  time  when  no  epidemic  of  the  disease  was  raging, 
and  by  doing  it  at  a  time  when  the  person  infected  was  in  the  most 
perfect  physical  condition,  the  dangers  of  the  malady  might  be 
mitigated. 

There  was  always  danger,  however,  that  the  induced  disease  being 
true  variola  might  prove  unexpectedly  severe,  or  even  fatal,  and 
that  each  inoculated  individual,  suffering  from  the  contagious  dis- 
ease, might  start  an  epidemic. 

2.  Jennerian  vaccination:  In  1791  a  country  schoolmaster  named 
Plett,  living  in  the  town  of  Starkendorf  near  Kiel  in  Germany, 
seems  to  have  made  the  first  endeavor  to  subject  the  oft-repeated 
observation,  that  persons  who  had  acquired  cow-pox  did  not  subse- 
quently become  infected  with  smallpox,  to  experimental  demon- 
stration, by  inserting  cow-pox  virus  into  three  children,  all  of  whom 
escaped  smallpox. 

The  father  of  vaccination,  and  the  man  to  whom  the  world  owes 
one  of  its  greatest  debts,  was  Edward  Jenner,  who  performed  his 
first  experiment  on  May  14,  1796,  when  he  transferred  some  of  the 
contents  of  a  cow-pox  pustule  on  the  arm  of  a  milkmaid  named 
Sarah  Nelmess  to  the  arm  of  a  boy  named  John  Phips.  After  the 
lad  had  recovered  from  the  experimental  cow-pox  thus  produced, 
he  subsequently  introduced  smallpox  pus  into  his  arm  and  found 
him  fully  immunized  and  insusceptible  to  the  disease.  This  led 
Jenner  to  perform  many  other  experiments,  and  record  his  ob- 
servations in  numerous  scientific  memoirs.  The  success  of  his 
work  immediately  attracted  the  attention  of  both  scientific  investi- 
gators and  sanitarians,  and  its  outcome  has  been  the  establishment 
of  compulsory  vaccination  by  legal  enactment  in  nearly  all  civilized 
countries,  with  the  result  that  smallpox,  instead  of  being  one  of  the 
most  prevalent  and  most  dreaded  diseases,  has  become  one  of  the 
most  rare  and  least  feared. 

The  immunity  acquired  through  vaccination  is  active  and  usually 
of  prolonged  duration.  It  is  subject  to  the  same  variations  ob- 
served in  other  experimentally  acquired  immunities,  these  varia- 
tions explaining  the  occasional  failures  which  constitute  the  "  stock 
in  trade"  of  those  who  still  remain  unconvinced  of  the  scientific 
basis  and  efficacy  of  the  procedure. 

Though  a  thorough  analysis  of  the  irregularities  and  exceptions 
of  vaccination  would  be  of  much  interest,  a  brief  mention  of  the 
most  important  must  suffice  for  the  present  argument. 

The  first  controversial  point  is  the  nature  of  the  " vaccine,"  or 
yirus  used  in  the  operation.  It  is  obtained  from  calves  or  heifers 
suffering  from  experimental  cow-pox,  and  is  a  virus  descended  from 


94  Immunity 

various  spontaneous  cases  of  cow-pox  observed  in  places  remote 
from  one  another.  Experts  are  undecided  whether  cow-pox  is 
variola  modified  by  passage  through  the  cow  so  that  the  transplanted 
micro-organisms  are  only  capable  of  inducing  a  local  instead  of  a 
general  disease,  or  whether  it  is  an  independent  affection  natural  to 
the  cow. 

In  reality  the  matter  is  unimportant,  so  long  as  the  desired  effect 
is  accomplished,  and  the  true  lineage  of  the  virus  is  only  a  matter 
of  scientific  curiosity.  As  immunity  is  almost  invariably  a  specific 
effect  resulting  from  infection,  it  would  seem  most  likely  that  cow- 
pox  and  smallpox  were  originally  identical. 

The  advantage  of  "vaccination"  over  " inoculation "  is  that  the 
induced  disease  is  local  and  not  dangerous  except  in  rare  cases, 
and  that  it  is  not  contagious.  The  natural  variations  in  the  sus- 
ceptibility of  different  vaccinated  individuals  determine  that  a  few 
persons  cannot  be  successfully  vaccinated,  being  immune  to  the 
mildly  invasive  organisms  of  vaccinia,  though  perhaps  susceptible 
to  the  actively  invasive  organisms  of  variola;  that  a  few  individuals 
shall  prove  abnormally  susceptible  to  vaccinia  so  that  the  disease 
departs  from  its  usual  local  type  and  generalizes,  but  that  in  nearly 
all  cases  the  disease  will  follow  the  well-known  type  of  a  local  lesion 
characterized  by  definite  periods  of  incubation,  vesiculation, 
pustulation,  and  cicatrization. 

The  occasional  variations  in  immunity  of  different  individuals 
also  determine  that  having  been  vaccinated  once  an  individual 
may  not  again  become  susceptible  to  vaccination,  though  he  may 
become  susceptible  to  the  more  actively  invasive  organisms  of  variola, 
or  that  he  may  soon  become  again  susceptible  to  both  diseases,  or 
that  in  very  rare  cases  no  immunity  against  variola  will  result  from 
vaccination.  In  most  cases  successful  vaccination  can  be  repeated 
once  or  twice  at  intervals  of  seven  or  ten  years,  and  experience 
shows  that  the  immunity  against  smallpox  conferred  by  vaccination 
is  of  longer  duration  and  usually  becomes  permanent  after 
vaccination  has  been  repeated  once  or  twice. 

Sanitarians  are  accustomed  to  speak  of  efficient  and  inefficient 
vaccination.  These  are  vague  terms  and  do  not  seem  to  be  under- 
stood by  the  laity.  Efficient  vaccination  is  vaccination  repeated 
as  often  as  is  necessary.  It  has  already  been  shown  that  individual 
variations  determine  that  a  few  individuals  never  become  immune, 
hence  never  can  be  efficiently  vaccinated.  Other  persons  are 
efficiently  vaccinated  by  a  single  operation.  The  term  is  usually 
interpreted  to  indicate  that  which  experience  has  shown  to  be 
efficient  in  average  cases. 

Failures  not  uncommonly  result  from  causes  having  nothing  to 
do  with  the  problems  of  immunity.  That  an  operation  of  scarifica- 
tion has  "been  performed  upon  a  child,  and  that  a  scar  has  remained 
thereafter  may  mean  nothing.  It  is  not  the  operation  but  the  dis- 


Vaccination  95 

ease  that  achieves  the"  result,  and  if  the  operation  be  improperly 
done,  poor — i.e.,  old  or  inert — matter  introduced,  or  if  after  intro- 
duction it  be  destroyed  by  the  application  of  antiseptics,  no  effect 
can  be  expected.  Hence  all  persons  that  have  been  vaccinated  may 
not  have  had  vaccinia,  the  essential  condition  leading  to  immunity. 
Nor  does  the  occurrence  of  a  local  lesion  act  as  a  guarantee  that 
vaccinia  has  been  induced.  Careful  examination  of  the  resulting 
lesions  should  always  be  made,  that  the  type  of  the  infection  may  be 
studied.  It  is  the  disease,  vaccinia,  that  must  occur — three  days' 
incubation,  three  days'  vesiculation,  three  days'  pustulation,  and 
subsequent  cicatrization  with  the  formation  of  a  punctate  scar. 

An  arm  may  be  made  very  sore,  may  suppurate  or  even  become 
gangrenous,  without  vaccinia  having  occurred  or  the  desired  benefit 
attained. 

The  accidents  of  vaccination  were  formerly  numerous  and  some- 
times disastrous  because  of  the  general  inattention  to  the  quality 
of  the  materials  used,  the  mode  of  inserting  them,  the  condition  of 
the  patient's  skin,  and  the  careless  treatment  of  the  resulting  lesions. 
When  human  virus  was  used,  that  is,  matter  taken  from  a  vaccinia 
lesion  from  a  human  being,  the  transmission  of  human  diseases, 
such  as  syphilis  and  erysipelas,  occasionally  took  place;  now  these 
are  rare  accidents  indeed,  because  no  virus  is  employed  except 
that  taken  from  carefully  selected  and  treated  calves  or  heifers. 
When  no  attention  was  paid  to  the  quality  of  the  bovine  virus,  and 
no  governmental  inspection  of  laboratories  required,  the  accidental 
contamination  of  the  virus  occasioned  a  small  number  of  accidental 
infections  of  the  wound.  There  are  a  good  many  cases  of  phlegmon, 
gangrene  and  tetanus  in  the  older  literature.  But  these  evils  are 
becoming  less  and  less  as  greater  attention  is  given  to  the  selection 
and  preparation  of  the  virus.  Some  accidents  and  some  few  deaths 
there  will  probably  always  be,  just  as  there  are  occasional  accidents 
and  occasional  fatal  results  following  all  kinds  of  trivial  injuries, 
though  care  will  eliminate  them  as  the  sources  of  accident  are  bet- 
ter understood. 

3.  Pasteurian  vaccination  or  bacterination:  Although  the  word 
vaccination  is  derived  from  the  Latin  vacca,  "a  cow,"  and  was  first 
employed  in  connection  with  Jenner's  method  of  introducing  virus 
modified  by  passage  through  a  cow,  Pasteur,  in  honor  of  Jenner, 
applied  it  to  every  kind  of  protective  inoculation,  and  the  word 
bacterination  is  only  introduced  for  the  purpose  of  indicating  certain 
differences  in  the  method. 

In  1880  Pasteur*  observed  that  some  hens  inoculated  with  a  cul- 
ture of  the  bacillus  of  chicken  cholera  that  had  been  on  hand  for 
some  time  did  not  die  as  was  expected.  Later,  securing  a  fresh  and 
virulent  culture,  these  and  other  chickens  were  inoculated.  The 
former  hens  did  not  die,  the  new  hens  did.  Quick  to  observe  and 
*  "Compte  rendu  de  la  Soc.  de  Biol.,  1880,  239;  315  et  seq. 


96  Immunity 

study  phenomena  of  this  kind,  he  investigated  and  found  that  when 
chickens  were  inoculated  with  old  and  non-virulent  cultures  they 
acquired  immunity  against  virulent  cultures.  This  led  him  to  the 
recommendation  of  the  employment  of  attenuated  cultures  as 
vaccines  against  the  disease,  and  to  the  achievement  of  great  success 
in  preventing  epidemics  by  which  great  numbers  of  the  barnyard 
fowls  of  France  were  being  destroyed. 

In  1 88 1  Pasteur,*  in  experimenting  with  Bacillus  anthracis,  ob- 
served that  if  the  organism  were  cultivated  at  unusually  high  tem- 
peratures it  lost  the  power  of  producing  spores,  and  diminished  in 
virulence.  He  also  found  that  when  the  organisms  had  been  so 
attenuated,  they  could  not  regain  virulence  without  artificial  manipu- 
lation. It  occurred  to  him  that  such  organisms,  possessing  feeble 
virulence,  might  be  able  to  confer  immunity  upon  animals  into  which 
they  were  inoculated,  and  he  continued  to  investigate  the  subject 
until  he  found  that  by  using  three  " vaccines"  or  modified  cultures 
of  increasing  virulence,  it  was  possible  to  render  animals  immune 
against  the  unmodified  organisms.  This  method  was  put  to  practical 
test  with  great  success,  and  has  since  been  extensively  practised 
in  different  parts  of  the  world. 

Arloing,  Cornevin  and  Thomas,  f  and  KittJ  found  that  exposure 
of  the  Bacillus  anthracis  symptomatici  to  a  high  temperature  in 
the  dry  state  modified  its  virulence  and  devised  a  practical  method 
of  protecting  cattle  against  symptomatic  anthrax  by  inoculating 
them  with  powdered  muscle  tissue  containing  the  bacilli  attenuated 
by  drying  and  exposure  to  85°C.  This  method  has  since  been  in  use 
in  many  countries,  and  has  given  excellent  satisfaction. 

In  1889  Pasteur, §  continuing  his  researches  upon  the  experimental 
modification  of  the  germs  of  disease  and  their  use  as  prophylactics, 
published  his  famous  work  upon  rabies,  and  showed  that,  although 
the  micro-organism  of  that  disease  had  so  far  eluded  discovery,  it 
was  contained  in  the  central  nervous  system  of  diseased  animals, 
where  it  could  be  modified  in  virulence  by  drying.  By  placing  spinal 
cords  removed  from  rabid  rabbits  in  a  glass  jar  containing  calcium 
chlorid,  he  was  able  to  diminish  the  virulence  of  the  contained 
micro-organisms  according  to  the  duration  of  the  exposure.  The 
introduction  of  the  attenuated  virus  was  followed  by  the  development 
of  a  certain  degree  of  immunity.  By  repeated  inoculation  of  more 
and  more  active  viruses  animals  acquired  complete  immunity  against 
street  virus.  These  experiments  formed  the  basis  of  the  "Pasteur 
method"  of  treating  rabies,  which  is  nothing  more  than  immuniza- 
tion with  the  modified  germs  of  the  disease  during  the  long  incubation 
period  of  the  disease. 

*  "Compte  rendu  de  la  Soc.  de  Biol.  de  Paris,"  1881,  xcn,  pp.  662-665. 
f  "Le  Charbon  Symptomatique  du  Boeuf,"  Paris,  1887. 
J"Centralbl.  f.  Bakt.,"  etc.,  i,  p.  684. 
§" Compte  rendu  de  la  Soc.  de  Biol.  de  Paris,"  1881,  cvm,  p.  1228. 


Immunity  Acquired  by  Intoxication  97 

Haffkine*  found  that  the  introduction  of  killed  cultures  of  virulent 
cholera  spirilla  produced  immunity  against  the  living  micro-organ- 
isms, and  used  the  method  with  considerable  success  for  preventing 
the  disease.  Laterf  he  applied  the  same  method,  also  with  consider- 
able success,  for  the  prevention  of  bubonic  plague,  and  A.  E.  WrightJ 
followed  pretty  much  the  same  method  for  the  prevention  of  typhoid 
fever. 

In  all  these  cases  the  immunity  induced  by  the  experimental 
manipulations  is  specific  in  nature,  and  variable  in  intensity,  ac- 
cording to  the  method  of  treatment  adopted  and  the  thoroughness 
with  which  it  is  carried  out. 

2.  Immunity  Acquired  by  Intoxication. — Bacterio- toxins  form  a 
miscellaneous  group  of  active  bodies  of  entirely  different  chemical 
composition  and  physiologic  activity.  Some  are  toxalbumins, 
some  are  enzymes,  some  are  bacterio-proteins.  The  true  nature  of 
the  greater  number  of  these  bodies  is  unknown,  but  study  of  their 
physiologic  action  has  brought  forth  the  important  fact  that  their 
behavior  toward  the  body  cells  is  in  no  way  different  from  the 
behavior  of  the  same  cells  toward  other  chemical  compounds  of 
similar  constitution,  and  that  nearly  all  physiologically  active  bodies 
introduced  into  living  organisms  produce  definite,  though  not 
necessarily  visible,  reactions. 

Such  reactions  are  now  known  as  antigenic,  and  the  substances 
by  which  they  are  induced  have  been  called  by  Deutsch  antigens. § 
Since  its  introduction  the  precise  meaning  given  the  word  by  Deutsch 
has  been  slightly  changed.  An  antigen  is  any  substance  which 
when  injected  into  the  body  of  a  living  organism  is  capable  of  pro- 
ducing a  chemicophysiologic  reaction  resulting  in  the  appearance  of 
a  neutralizing,  precipitating,  agglutinating,  dissolving,  or  other- 
wise antagonizing  substance  known  as  an  antibody. 

The  antigens  are,  so  far  as  known,  all  colloidal  substances.  They 
may  be  harmful  or  harmless,  active  or  inert,  living  or  dead,  organized 
or  unorganized.  The  reactions  are  specific  and  the  antibody  has 
specific  affinity  for  that  antigen  alone  by  which  its  formation  has 
been  excited. 

All  poisonous  substances  are  not  antigens,  even  though  a  certain 
immunity — in  the  sense  of  habituation  or  tolerance — may  follow 
their  repeated  administration.  One  may  become  habituated  or 
tolerant  to  a  certain  quantity  of  mercury  or  arsenic,  and  to  certain 
alkaloids,  such  as  morphin,  caffein,  nicotin,  cocain,  etc.,  but  he 
does  not  react  as  to  them  as  to  antigens  and  no  antibodies  an- 
tagonistic to  them  are  formed.  To  these  various  substances  he 
really  acquires  only  a  slight  degree  of  tolerance;  to  the  effects  of 

*  "Brit.  Med.  Jour.,"  1891,  n,  p.  1278. 

t  "  Brit.  Med.  Jour.,"  1895,  n,  p.  1541. 

j  Ibid.,  Jan.  30,  1897,  I,  p.  256. 

§  Deutsch  und  Feistmantel,  "  Die  Impfstoffe  und  Sera,"  1903,  Leipzig,  Thieme. 


98  Immunity 

injurious  antigens  he  may  acquire  an  almost  unlimited  degree  of 
immunity  through  the  formation  of  the  antibodies. 

From  remote  antiquity  it  has  been  known  that  those  who  regularly 
consume  small  quantities  of  poisons  become  irresponsive  to  their 
action,  and  it  is  well  known  that  Mithridates  attempted  this  mode  of 
defending  himself  from  his  enemies. 

Chauveau*  believed  that  the  immunity  conferred  by  inoculations 
of  bacteria  was  due  to  the  presence  of  their  soluble  products,  but  the 
first  direct  demonstration  of  the  fact  was  by  Salmon  and  Smith,  f  who, 
as  early  as  1886,  showed  that  it  was  possible  to  immunize  pigeons 
against  the  hog-cholera  bacillus  by  means  of  repeated  injection 
with  cultures  exposed  to  6o°C.,  and  containing  no  living  organisms. 
CharrinJ  found  it  possible  to  immunize  rabbits  against  Bacillus 
pyocyaneus  by  injecting  them  with  the  filtered  products  of  cultures 
of  that  organism,  and  Bonome§  similarly  to  immunize  animals 
against  Bacillus  proteus,  B.  cholera  gallinarum  and  the  pneu- 
mococcus.  Roux  and  Chamberland||  and  Roux**  were  able  by  the 
use  of  boiled  cultures  of  the  bacilli  of  malignant  edema,  and  of 
quarter  evil,  similarly  to  immunize  animals  against  these  respective 
infections. 

The  subject  was  much  further  elaborated  by  Roux  and  Yersmff 
in  their  experiments  with  diphtheria  toxin;  by  BehringJJ  in  his 
early  studies  of  diphtheria,  and  by  Kitasato§§  in  his  experiments 
with  tetanus. 

These  early  experiments  opened  a  wide  field,  through  the  investiga- 
tion of  which  we  now  know  that  the  products  as  well  as  the  living  or 
dead  bacteria  of  most  of  the  infectious  diseases,  when  properly 
introduced  into  animals,  can  induce  immunity. 

(B)  Passive  Acquired  Immunity. — Passive  immunity  is  always 
acquired,  never  natural.  It  depends  upon  defensive  factors  not 
originating  in  the  animal  protected,  but  artificially  or  experimentally 
supplied  to  it.  The  fundamental  principle  is  simple  and  has  become 
the  basis  of  serum  therapeutics.  If  the  immunized  animal  generates 
factors  by  which  the  infecting  bacteria  can  be  destroyed  or  the 
activity  of  their  products  overcome  in  its  body,  cannot  these  factors 
be  removed  and  the  benefit  they  confer  transferred  to  another 
animal  ? 

The  first  experiments  in  this  direction  seem  to  have  been  made 
by  Babes  and  Lepp,||||  who  found  that  the  blood-serum  of  animals 

*"Ann.  de  1'Inst.  Pasteur,"  1888,  2. 

t"Centralbl.  f.  Bakt.,"  etc.,  1887,  n,  No.  18,  p.  543. 

J  "Compte  rendu,"  de  la  Soc.  de  Biol.,  cv,  p.  756. 

§  "Zeitschrift  f.  Hyg.,"  v,  p.  415. 

j|  "Ann.  de  1'Inst.  Pasteur,"  1887,  12. 
>*Ibid.,  1888,  2. 
ft  Ibid.,  1888,  n,  p.  269. 
"|  "Deutsche  med.  Wochenschrift,"  1890,  No.  50. 

"  "Zeitschrift  fur  Hygiene,"  1891,  x,  p.  267. 
"Annales  de  1'Inst.  Pasteur,"  1889,  vol.  in. 


Passive  Acquired  Immunity  99 

immunized  to  rabies  showed  a  defensive  power  when  injected  into 
other  animals.  Ogata  and  Jasuhara*  found  that  the  subcutaneous 
injection  of  blood-serum  from  an  animal  immunized  against  anthrax 
enabled  the  injected  animals  successfully  to  resist  infection.  Behring 
and  Kitasatof  found  that  the  blood-serums  of  animals  immunized 
against  diphtheria  and  tetanus,  when  mixed  with  cultures  of  these 
respective  bacilli,  neutralized  their  power  to  produce  disease. 
KitasatoJ  found  that  if  mice  were  inoculated  with  tetanus  bacilli, 
they  could  be  saved  from  the  fatal  infection  by  the  intra-abdominal 
injection  of  some  blood-serum  from  a  mouse  immunized  against 
tetanus,  even  after  symptoms  of  the  disease  had  appeared.  Ehrlich§ 
showed  that  the  blood-serums  of  animals  immunized  against  abrin 
and  ricin  could  save  other  animals  from  the  fatal  effects  of  these 
respective  toxalbumins;  Phisalix  and  Bertrand,j|  and,  later,  Cal- 
mette**  found  the  blood-serum  of  animals,  immunized  against  the 
venoms  of  serpents,  similarly  possessed  the  power  of  neutralizing 
the  poisonous  effects  of  the  venoms.  Kosselff  found  that  the  blood- 
serum  of  animals,  immunized  against  the  poisonous  blood-serum  of 
eels,  contained, a  body  which  destroyed  or  neutralized  the  effects 
of  the  eels'  serum. 

Thus,  it  is  shown  that  in  each  case  in  which  defensive  reactions 
are  stimulated  in  experiment  animals,  the  reactions  are  accompanied 
by  the  appearance  in  the  blood-serum  of  those  animals  of  factors 
that  can  be  utilized  to  defend  other  animals  in  whose  bodies  no 
similar  reactions  have  taken  place. 

Passive  immunity  may  also  be  brought  about  in  a  few  cases 
by  the  injection  into  the  intoxicated  animal  of  substances,  other 
than  immunity  products,  that  have  a  specific  affinity  for  the  poison. 
Thus  Wassermann  and  TakakiJt  found  that  when  the  crushed  spinal 
cord  of  a  rabbit  was  mixed  in  vitro  with  tetanus  toxin,  the  poison 
was  quickly  absorbed  by  the  nerve-cells,  so  that  the  mixture  became 
inert  and  could  be  injected  into  animals  without  harm.  Wasser- 
mann also  found  that  the  same  effects  could  be  produced  in  the 
bodies  of  animals,  and  that  when  the  crushed  spinal  cord  was 
injected  into  an  animal  a  few  hours  previously,  or  a  few  hours  after 
a  fatal  dose  of  tetanus  toxin,  enough  of  the  combining  elements  re- 
mained in  the  blood  to  fix  the  toxin  before  it  anchored  itself  to  the 
central  nervous  system  of  the  intoxicated  animal.  Myers §§  found 
that  the  ground-up  tissue  of  the  adrenal  bodies  was  able  to  fix  and 
thus  annul  the  poisonous  effects  of  cobra  venom  in  vitro. 

'Centralbl.  f.  Bakt,"  etc.,  1890,  EX,  p.  25. 
'Deutsche  med.  Woch.,"  1890,  No.  49. 
'Zeitschrift  fiir  Hygiene,"  1892,  xn,  p.  256. 
'Deutsche  med.  Wochenschrift,"  1891,  Nos.  32  and  44. 
'Compte  rendu  Acad.  des  Sciences  de  Paris,"  cxvm,  p.  556. 
'Ann.  de  FInst.  Pasteur,"  1894,  vm,  p.  275. 


tt 


'Berliner  klin.  Woch.,"  1898,  p.  152. 
'Berliner  klin.  Wochenschrift,"  Jan.  3,  iJ 
'Lancet,"  July  2,  1898. 


ioo  Immunity 

In  all  these  cases  the  neutralizing  effects  are  either  accomplished 
or  initiated  by  factors  prepared  experimentally,  and  forced  upon 
the  animal  in  whose  body  their  activities  are  manifested. 


EXPERIMENTAL  INVESTIGATION  OF  THE  PROBLEMS  OF  IMMUNITY 

Very  important  contributions  were  made  by  Ehrlich,*  in  his 
work  upon  the  vegetable  toxalbumins,  ricin,  abrin,  and  robin, 
that  were  found  to  be  antigens  capable  of  producing  anti-ricin, 
anti-abrin  and  anti-robin  respectively,  each  antibody  being  capable 
of  neutralizing  the  effect  of  its  specific  antigen.  Kosself  investigated 
the  reactions  produced  by  toxic  eels'  blood  and  found  that  im- 
munity could  be  established  against  their  hemolytic  action,  and  that 
specific  antibodies  were  formed.  Phisalix  and  BertrandJ  showed 
that  immunity  could  also  be  produced  in  guinea-pigs  against  the 
action  of  viper  venom,  and  that  a  specific  antibody,  "antivenene" 
was  the  source  of  the  immunity. 

The  investigation  of  other  active  bodies  was  soon  begun.  In 
1893  Hildebrand§  studied  emulsin  and  found  that  it  produced  a 
definite  reaction  with  the  formation,  in  animals  injected,  of  an  anti- 
emulsin.  v.  Dlingern||  studied  proteolytic  enzymes  of  various 
bacteria,  and  showed  that  when  gelatin-dissolving  enzymes  were 
repeatedly  injected  into  animals,  definite  reactions  took  place, 
and  in  the  serum  a  body  appeared  that  inhibited  the  action  of  the 
ferment  in  a  test-tube.  Gheorghiewski**  immunized  animals  to 
cultures  of  Bacillus  pyocyaneus,  and  found  that  the  reaction  pro- 
voked caused  the  appearance  in  the  serum  of  some  body  that  pre- 
vented the  formation  of  the  blue  pigment  so  characteristic  of  the 
organism.  Morgenrothff  applied  the  same  principle  to  rennet, 
finding  that  it  produced  definite  reactions,  with  the  formation  of 
an  antibody  inhibiting  the  coagulation  of  milk.  Bordet  and 
Gengoujf  found  that  the  fibrin  ferment  of  the  blood  of  one  animal 
was  active  in  the  body  of  another  animal,  producing  an  inhibiting 
substance  by  which  the  coagulation  of  the  blood  of  the  first  animal 
could  be  delayed. 

The  studies  of  Kraus§§  showed  a  new  fact,  that  when  filtered  cul- 
tures of  the  cholera  spirillum  were  introduced  into  animals,  the 
serum  of  these  animals,  added  to  the  filtered  culture  in  a  test-tube, 
caused  the  appearance  of  a  delicate  flocculent  precipitate,  specific 
precipitate. 

*  "Deutsche  med.  Woch.,"  1891,  Nos.  32  and  44. 

f  "Berliner  klin  Wochenschrift,"  1898. 

j  Atti  d  XI  Congr.  med.  internaz.  Roma,  1894,  11,  200-202. 

§  "Virchow's  Archives,"  Bd.  cxxxi. 

I]  "  Miinchener  med.  Woch.,"  Aug.  15,  1898. 
'*  "Ann.  de  1'Inst.  Pasteur,"  1899. 
ft  "  Centralbl.  f.  Bakt.,"  etc.,  1899,  xxvi,  p.  349. 
JJ  "Ann.  de  1'Inst.  Pasteur,"  1903,  xvn,  p.  822. 
§§  "Wien.  klin.  Woch.,"  1897. 


Experimental  Investigation  of  the  Problems  of  Immunity  101 

Wassermann  and  Schiitze*  found  that  when  cow's  milk  was; 
repeatedly  injected  into  rabbits,  their  serum  acquired  the  property 
of  occasioning  a  precipitate  when  added  to  cows'  milk,  but  not  when 
added  to  goats'  or  any  other  milk.  If,  however,  the  rabbit  had  been 
repeatedly  injected  with  goats'  milk  or  human  milk,  its  serum  would 
precipitate  with  those  milks  respectively,  and  not  with  cow's  milk. 
The  reaction  was  thus  shown  to  be  specific. 

Myers f  found  that  the  repeated  intraperitoneal  injection  of 
egg-albumen  into  rabbits  caused  their  serum  to  give  a  dense  pre- 
cipitate when  added  to  solutions  of  egg-albumen. 

Tchisto  witch  t  found  that  eels'  serum  injected  into  animals 
produced  a  reaction  in  which  immunity  to  its  poisonous  action  was 
associated  with  the  ability  of  their  serum  to  produce  a  precipitate 
when  added  to  the  eels'  serum. 

Closely  connected  with  these  various  reactions  are  certain  others 
variously  spoken  of  as  cytotoxic,  cytolytic,  hemolytic,  bacteriolytic, 
etc.  The  first  observation  bearing  upon  these  was  made  by  R. 
Pfeiffer,§  who  found  that  when  guinea-pigs  received  frequent 
intraperitoneal  injections  of  cholera  spirilla  and  became  thoroughly 
immunized,  their  serum  behaved  very  peculiarly  toward  the  bacteria 
in  the  peritoneal  cavity  of  freshly  infected  animals,  in  that  it  caused 
them  to  become  aggregated  into  granular  masses  and  subsequently 
to  disappear.  This  became  known  as  "Pfeiffer's  phenomenon." 
The  serum  of  the  immunized  animal  was  devoid  of  action  by  itself, 
the  serum  of  the  infected  animal  was  inactive,  but  the  combination 
of  the  two  brought  about  dissolution  of  the  micro-organisms.  Later 
it  was  shown  by  MetschnikofT||  that  the  living  animal  was  not  a  factor 
in  the  process,  but  that  what  was  seen  in  the  peritoneal  cavity  could 
be  reproduced  in  a  test-tube,  though  not  quite  as  well. 

Bordet**  made  frequent  injections  of  defibrinated  rabbits'  blood 
into  guinea-pigs,  and  obtained  a  serum  that  had  a  solvent  action 
upon  the  rabbit's  corpuscles  in  vitro,  and  showed  that  the  induced 
hemolysis  resembled  in  all  points  the  bacteriolysis. 

Ehrlichf  f  and  Morgenroth  studied  the  hemolytic  action  of  the 
serum  of  goats  that  had  been  frequently  injected  with  the  de- 
fibrinated blood  of  sheep  and  goats,  and  were  able  to  point  out  the 
mechanism  of  the  corpuscle  solution  or  hemolysis.  It  was  found 
to  depend  upon  two  associated  factors,  one  of  which,  the  lysin  or 
solvent,  was  present  in  normal  blood,  and  was  called  "addiment" 
or  "complement"  and  another  present  only  in  the  serum  of  the 
reactive  animals,  called  the  "immune  body"  or  "intermediate  body." 
The  former  was  labile  and  easily  destroyed  by  heat,  the  latter 

*  "Deutsche  med.  Woch.,"  1900. 

f  "Lancet,"  1900,  n. 

j  "Ann.  de  1'Inst.  Pasteur,"  vol.  XIIT,  406. 

§  "Deutsche  med.  Wochenschrift,"  1896,  No.  7. 

||  "Ann.  de  1'Inst.  Pasteur,"  1895. 
**  Ibid.,  1898,  XTI. 
ft  "Berliner  klin.  Wochenschrift,"  1899. 


IO2  Immunity 

stabile  and  not  affected  by  heat  up  to  the  point  of  coagulation.  The 
experiments  were  confirmed  by  von  Diingern  and  many  others. 
It  is  to  be  observed  in  passing  that  this  reaction  differs  from  the 
direct  solution  of  the  corpuscles  in  vitro  by  cobralysin,  which  was 
studied  by  Myers,*  and  tetanolysin,  studied  by  Madsen,f  in  that  it 
is  intermediate,  and  only  brought  about  by  the  cooperation  of  two 
factors,  while  the  action  of  the  lysins  of  venom,  the  tetanus  bacillus, 
the  streptococcus,  Bacillus  pyocyaneus,  and  other  micro-organisms, 
is  direct  and  immediate. 

Myers  found,  however,  that  the  hemolytic  substance  of  venom, 
and  Madsen  that  the  hemolytic  products  of  Bacillus  tetani,  also 
produce  reactions  in  animals,  and  that  when  successful  immuniza- 
tion against  them  was  accomplished,  the  serums  of  the  experiment 
animals  became  antidotal  or  inhibiting  to  the  action  of  the  respective 
lysins. 

Von   Diingern {   found   that  by  injecting   dissociated   epithelial 
cells  from  the  trachea  of  oxen  into  the  peritoneal  cavity  of  guinea- 
pigs,  it  was  possible  to  produce  epitheliolysins;  Lindemann,§  that 
emulsions  of  kidney  substance  injected  into  animals  caused  them  to 
form  nephrolysins  or  nephrotoxins;  Landsteiner||  and  Metschnikoff ** 
in  the  same  manner  successfully  prepared  spermatoxin  by  injectin- 
the  spermatozoa  of  one  animal  into  the  peritoneal  cavity  of  anothe 
Metalnikoffff  found  that  if  he  introduced  the  spermatozoa  oK 
guinea-pig  into  the  peritoneum  of  another,  the  sperma  toxic  serum 
produced  was  solvent  for  the  spermatozoa  of  both.     Both  Metsch- 
nikoff  and   Metalnikoff  also  found   that   the   spermatoxin    when 
introduced  into  animals  was  active  in  producing  anti-sperma  toxin 
by  which  the  destructive  action  of  the  serum  upon  spermatozoa 
could  be  inhibited. 

Metschnikoff  Jt  and  Funck§§  found  that  animals  treated  with 
emulsions  of  the  spleen,  and  mesenteric  lymph-nodes  of  one  kind  of 
animal,  produced  sera  whose  action  was  agglutinative  and  solvent 
for  leukocytes  and  lymph-cells.  Delezene||  ||  found  that  dissociated 
liver  cells  injected  into  animals  similarly  caused  the  formation  of  a 
specific  cytotoxic  serum. 

All  of  these  reactions  are  indirect  and  intermediate,  and  take 
place  under  appropriate  conditions  both  in  the  bodies  of  animals 
and  in  the  test-tube. 

Thus  the  number  of  antigenic  reactions  that  can  be  brought 
about  in  the  bodies  of  animals  seems  to  be  limitless,  and,  strange 

*  "Trans.  Path.  Soc.  of  London,"  LI. 

t  "Zeitschr.  f.  Hyg.,"  1899,  xxxm,  p.  239. 

j  "Munchener  med.  Wochenschrift,"  1899. 

§  "Ann.  de  1'Inst.  Pasteur,"  1900. 

||  "Centralbl.  f.  Bakt.,"  etc.,  1899,  xxv. 
**  "Ann.  de  1'Inst.  Pasteur,"  1899. 

ft  Ibid.,  1900.  JJ  Ibid.,  1899. 

"§  "Centralbl.  f.  Bakt.,"  etc.,  1900,  xxvu. 

||  "Compte  rendu  de  1'Acad.  des  Sciences,"  1900,  cxxx,  pp.  938,  1488. 


Allergia  or  Anaphylaxis  103 

as  it  may  seem,  the  antibodies  produced  in  the  body  of  one  animal 
may  act  as  antigens  when  introduced  into  another.  Thus,  Ehrlich 
and  Morgenroth  in  their  studies  of  hemolysis  found  that  serums 
rich  in  immune  bodies  produced  reactions  yielding  anti-immune 
bodies,  which  inhibited  the  activities  of  the  respective  immune 
bodies  by  whose  stimulation  they  were  produced. 

The  reactions  which  when  repeated  may  lead  to  immunity 
and  to  the  formation  of  antibodies  seem  to  be  followed  by  con- 
stitutional disturbances  much  more  profound  than  would  be  sup- 
posed from  the  apparent  freedom  from  symptoms  manifested  by 
the  animal.  As  early  as  1839  Magendie  observed  that  if  a  rabbit 
was  given  an  injection  of  albumin,  and  then,  some  days  later,  a 
second  injection,  it  was  made  very  ill  and  might  die.  About  1900 
Mattson  in  private  conversation  called  the  author's  attention  to  the 
fact  that  when  guinea-pigs  used  for  testing  antitoxic  serums  were 
subsequently  injected  with  another  dose  of  serum,  they  commonly 
died.  Not  being  understood,  the  matter  was  not  thought  worthy 
of  publication.  Otto*  speaks  of  this  fatal  action  of  serums  as  the 
"Theobald-Smith  phenomenon,"  the  fact  having  first  been  pointed 
out  to  him  by  Smith. 

The  first  to  realize  the  importance  of  the  condition  seem  to  have 

<en  Portier  and  Richet,f  who  studied  the  effect  of  extracts  of  the 
k  .  isonous  tentacles  of  actiniens  upon  dogs  which  were  found  to  die 
iiiore  quickly  and  from  smaller  doses  given  at  a  second  injection 
than  at  the  first.  To  this  increase  of  sensitivity  to  the  poison  brought 
about  by  the  initial  dose  they  gave  the  name  anaphylaxis  (av  nega- 
tive, <pv\a%is  protection,  destroying  protection  or  breaking  down  the 
defenses). 

The  therapeutic  employment  of  diphtheria  antitoxic  serum 
was  scarcely  popularized  before  the  medical  profession  was  shocked 
by  the  sudden  death  of  the  healthy  child  of  a  noted  German  pro- 
fessor after  a  prophylactic  injection,  and  in  1896  Gottsteinf  was 
able  to  collect  eight  deaths  following  the  use  of  the  serum, four  of  them 
being  persons  not  ill  with  diphtheria,  von  Pirquet  and  Schick§  also 
pointed  out  that  in  a  certain  proportion  of  cases  the  injection  of 
horse-serum  in  man  is  followed  by  urticarial  eruptions,  joint-pains, 
fever,  swelling  of  the  lymph-nodes,  edema  and  albuminuria,  these, 
symptoms  usually  appearing  after  an  incubation  period  of  eight  to 
thirteen  days,  and  constituting  what  they  call  the  "  serum  disease," 
or  alter gia.  Sometimes  these  reactions  are  immediate;  sometimes 
death  appears  imminent,  and,  as  has  been  observed,  death  some- 
times occurs. 

The  investigation  of  the  subject  was  taken  up  in  1905  by  Rosenau 

*  von  Lenthold,  ".Gedenkschrift,"  Bd.  i,  pp.  9,  16,  18. 
t  "  Compte  rendu  de  la  Soc.  de  Biol.  de  Paris,"  1902. 
J  "Therap.  Monatschrift,"  1896. 
§  "Die  Serumkrankheit,"  Leipzig  and  Wien,  1905. 


104  Immunity 

and  Anderson,*  who  pursued  it  with  great  interest  and  industry, 
by  Gay,f  Gay  and  Southard,  J  and  others. 

Experimental  study  shows  that  when  an  animal  is  injected 
with  an  alien  protein  of  almost  any  kind,  a  reaction  takes  place 
that  usually  is  not  completed  under  six  days.  If  a  second  injec- 
tion is  given  before  the  reaction  is  perfected,  the  mechanism  of 
immunity  is  set  in  action,  and  the  animal  proceeds  to  defend  itself 
through  the  various  means  described.  If  the  second  administration 
be  deferred,  however,  until  the  first  reaction  is  completed,  it  seems 
to  find  the  animal  in  a  state  of  disturbed  biologic  equilibrium,  the 
nature  of  which  is  not  understood,  but  which  is  characterized  by  a 
profound  disturbance  that  may  terminate  in  death.  The  reaction 
is  quite  specific;  the  sensitization,  once  effected,  may  continue 
throughout  the  remainder  of  the  life  of  the  animal  and  be  trans- 
mitted from  the  mother  to  her  offspring  through  her  blood.  The 
reaction  can  be  brought  about  by  feeding  the  protein  or  by  injecting 
it.  It  has  an  important  bearing  upon  infection  and  immunity,  the 
chief  example  being  seen  in  the  tuberculin  reaction. 

The  symptomatology  of  anaphylaxis  is  interesting  and  char- 
acteristic. When  it  is  desirable  to  study  it,  a  guinea-pig  is  first 
given  a  sensitizing  dose  of  horse-serum.  This  may  be  very  small. 
Rosenau  and  Anderson  found  one  guinea-pig  to  be  sensitized  by 
one-millionth  of  a  cubic  centimeter.  In  most  of  their  work  they  used 
less  than  3^50  cc.  It  is  necessary  to  wait  until  the  effects  of  this 
first  injection  are  completely  over  before  giving  the  poisoning  dose. 
This  period  of  incubation  lasts  about  twelve  days.  After  the  lapse 
of  this  time,  the  second  dose,  usually  about  Jf  o  cc.,  is  given.  Both 
doses  are  given  by  injection  into  the  peritoneal  cavity. 

The  symptoms  come  on  almost  immediately  after  the  second 
dose.  The  animal  is  profoundly  depressed,  extremely  uneasy,  pants 
for  breath,  and  suffers  from  intense  itching  of  the  face.  It  soon 
falls,  continues  to  gasp  for  breath,  and  dies  within  an  hour.  The 
disturbances  in  the  body  of  the  animal  are  sufficient  to  account  for 
the  symptoms.  Extensive  lesions  exist,  the  first  to  be  described 
by  Rosenau  §  affecting  the  mucous  membrane  of  the  stomach,  which 
appeared  ecchymotic  and  ulcerated.  Gay  and  Southard  ||  found 
hemorrhages  in  most  of  the  organs,  and  believe  anaphylaxis  to 
depend  upon  the  presence,  in  the  blood  of  the  sensitized  animal,  of 
a  substance  to  which  they  have  given  the  name  anaphylactin.  Bes- 
redka  and  Steinhardt**  found  that  by  the  repeated  injection  of 

*  "Journal  of  Medical  Research,"  1906,  xv,  p.  207;  "Bull.  No.  29  of  the 
Hygienic  Laboratory,"  Washington,  D.  C.,  1906;  "Bull.  No.  36,"  1907,  Ibid.; 
"Jour.  Med.  Research,"  1907, xvi,  No.  3,  p.  381;  "Jour.  Infectious  Diseases," 
1907,  iv,  No.  i,  p.  i,  "Jour.  Infectious  Diseases,"  1907,  vol.  rv,  p.  552. 

t  "Jour.  Med.  Research,"  May,  1907,  xvi,  No.  2,  p.  143. 

t  Ibid.,  June,  1908,  xvm,  No.  3,  p.  385. 

§"Bull.  No.  32  of  the  Hygienic  Laboratory/'  Washington,  D.  C.,  October, 
1906. 

||  "Jour.  Med.  Research,"  July,  1908,  xrx,  No.  i,  pp.  i,  5,  17. 

**  "Ann.  de  1'Inst.  Pasteur,"  February  25,  i9O7,xxi,  No.  2,  pp.  117-127. 


Explanation  of  Immunity  105 

horse-serum  into  guinea-pigs,  the  intervals  being  too  short  to  permit 
anaphylaxis,  antianaphylactin  could  be  prepared.  It  seems  difficult, 
however,  to  imagine  how  such  a  substance  could  remain  in  the  blood 
throughout  the  entire  subsequent  life  of  the  animal. 

Vaughan  has  endeavored  to  explain  anaphylaxis  by  assum- 
ing that  when  the  strange  protein  in  the  blood  reaches  the  cells 
it  is  slowly  broken  down  by  enzymic  action,  but  that  the  cells, 
having  once  acquired  the  property  of  destroying  it,  seize  eagerly 
upon  the  protein  the  next  time  it  is  offered,  disintegrate  it  rapidly, 
and  so  disseminate  throughout  the  body  the  degradation  products, 
some  of  which  may  be  toxic  and  account  for  the  reaction. 

Anaphylaxis  is  not  a  disturbance  of  the  cells  of  the  body,  as 
some  have  thought,  but  is  at  least  in  part  a  disturbance  of  the 
composition  of  the  blood,  as  can  be  shown  by  the  occurrence  of 
what  is  known  as  passive  anaphylaxis.  If  the  blood-serum  of  a 
sensitized  animal  be  withdrawn  and  injected  into  a  normal  animal 
of  the  same  kind,  it  carries  the  sensitization  with  it.  The  new 
animal,  however,  does  not  become  sensitized  at  once,  but  only 
after  some  days,  hence  it  is  equally  true  that  the  disturbance  is 
not  solely  in  the  blood,  else  why  should  not  the  sensitization  be 
immediately  present  upon  the  injection  of  the  serum? 

Anaphylaxis  may,  furthermore,  be  local.  Thus,  when  certain 
substances  like  tuberculin  are  dropped  in  the  eye  there  is  no  effect, 
but  when  a  second  application  is  made,  after  some  weeks,  the  eye 
may  be  reddened. 

Anaphylaxis  may  play  a  role  in  infection.  In  cases  where  an 
attack  of  an  infectious  disease  leaves  no  immunity,  the  body  may 
be  left  hypersensitive  to  subsequent  attacks. 

EXPLANATION  OF  IMMUNITY 

Before  the  facts  now  at  our  disposal  had  been  gathered  together, 
and  before  the  phenomena  of  immunity  against  infection  had  been 
compared  with  those  of  intoxication,  Pasteur*  and  Klebsf  en- 
deavored to  explain  acquired  immunity  by  supposing  that  micro- 
organisms living  in  the  infected  animal  used  up  some  substance 
essential  to  their  existence,  and  so  died  out,  leaving  the  soil  unfit 
for  further  occupation.  This  was  known  as  the  "  exhaustion 
theory."  WernichJ  and  Chauveau§  thought  it  more  probable 
that  the  micro-organisms  after  having  lived  in  the  body  left 
behind  them  some  substance  inimical  to  their  further  existence. 
This  was  known  as  the  "retention  theory."  These  hypotheses  are 
of  historic  interest  only,  and  deserve  no  more  than  passing  men- 
tion, as  they  both  fail  to  explain  natural  immunity  or  immunity 
against  intoxication. 

*  "  Compte  rendu  de  la  Soc.  de  Biol.  de  Paris,"  xci. 

t  "Arch.  f.  experimentelle  Path.  u.  Pharmak.,"  xm. 

|  "Virchow's  Archives,"  Bd.  LXXVIII. 

§  "  Compte  rendu  de  la  Soc.  de  Biol.  de  Paris,"  xc  and  xci. 


io6 


Immunity 


Karl  Roser*  observed  that  the  leukocytes  of  the  bodies  of  higher 
animals  sometimes  enclosed  bacteria  in  their  cytoplasm.  Koch, 
Sternberg,  and  others,  confirmed  the  observation,  but  no  attention 
was  paid  to  it  until  Metschnikofff  correlated  it  with  other  known 
facts  and  original  observations,  and  came  to  the  conclusion  that  the 
enclosed  bacteria  had  been  eaten  by  the  leukocytes  in  which  they 
were  killed  and  digested,  and  that  the  behavior  of  the  cells  toward 
the  bacteria  afforded  an  explanation  of  the  mechanism  by  which 
recovery  from  the  infectious  diseases  takes  place.  The  original 
conception  upon  which  this  "theory  of  phagocytosis"  was  founded, 
refers  recovery  in  many,  if  not  all  of  the  infectious  diseases,  to  the 
successful  destruction  of  the  invading  bacteria  by  the  body  cells, 
especially  the  leukocytes.  These  devouring  cells  Metschnikoff 
called  phagocytes,  and  of  them  he  recognized  two  classes,  the  micro- 
phages,  which  are  white  blood-corpuscles,  and  the  macrophages, 
which  are  larger  cells  derived  from  the  endothelial  and  other  tissues. 


Fig.  17. — Phagocytosis;  the  omentum  immediately  after  injection  of  typhoid 
bacilli  into  a  rabbit.  Meshwork  showing  a  macrophage,  intermediate  forms 
and  a  trailer,  all  containing  intact  bacilli  (Buxton  and  Torry). 

Metschnikoff,  his  associates,  and  his  pupils  soon  collected  evidence 
sufficient  to  show  that  phagocytosis,  if  not  the  chief  factor  in  de- 
fending the  body  from  infectious  organisms,  is  at  least  an  important 
one.  Many  of  the  most  interesting  facts  are  described  in 
Metschnikoff 's  books,  "Etudes  sur  1' Inflammation"  and  "Im- 
munite  dans  les  Maladies  Infectieuses,"  which  every  interested 
student  of  the  subject  should  read. 

These  studies  show  that  in  nearly  all  cases  in  which  animals  are 
naturally  immune  against  infection,  the  leukocytes  are  active  in 
their  phagocytic  behavior  toward  them;  that  in  acquired  immunity, 
the  leukocytes  previously  inactive,  become  active  toward  them; 

*"Beitrage  zur  Biologic  niederster  Organismen,"  Inaugural  Dissertation, 
Marburg,  1881. 

f'Virchbw's  Archives,"  Bd.  xcvi,  p.  177;  "Ann.  de  Plnst.  Pasteur,"  1887, 
t.  i,  p.  321. 


Phagocytosis — Opsonins  107 

that  the  enclosure  of  bacteria  within  the  cells  sometimes  results  in 
the  death  of  the  cells,  sometimes  in  the  death  of  the  bacteria;  that 
phagocytosis  is  much  more  active  in  diseases  in  which  the  bacteria 
have  limited  toxicogenic  powers,  and  in  which  they  probably  exert 
a  positively  chemotactic  influence  upon  the  cells,  than  in  cases  in 
which  the  bacteria  are  strongly  toxicogenic  and  probably  exert  an 
injurious  and  negatively  chemotactic  influence  upon  them,  and 
that  when  the  toxicogenic  power  of  the  bacteria  is  great,  many  of 
the  phagocytes  are  killed  and  dissolved — phagolysis.  Study  of 
the  primitive  forms  of  animal  life  shows  that  amebae  constantly 
feed  upon  smaller  organisms,  some  almost  exclusively  upon  bacteria, 
which  they  are  able  to  kill  and  digest  through  an  intracellular 
enzyme  demonstrated  by  Mouton,*  and  called  amebadiastase,  and 
regarded  as  a  form  of  trypsin.  The  intracellular  digestion  of 
ccelenterate  animals  is  accomplished  by  means  of  actinodiastase,  an 
enzyme  discovered  by  Fredericq,  and  studied  by  Mesnil.  It  seems 
to  be  related  to  papine  and  digests  albuminoids.  The  digestion  of 
erythrocytes  and  tissue  fragments  is  accomplished  through  an 
enzyme  of  the  macrophages,  which  Metschnikoff  calls  macrocytase, 
that  of  bacteria  through  an  enzyme  of  the  microphages,  which  he 
calls  microcytase.  In  phagolysis  these  respective  ferments  are 
liberated  into  the  plasma,  imparting  to  it  a  bactericidal  and  bacterio- 
ly tic  action  similar  to  that  normally  peculiar  to  the  cytoplasm  of  the 
cells.  The  dissemination  of  the  enzymes  in  phagolysis,  with  re- 
sulting bacteriolytic  power  of  the  blood  plasma  and  serum,  is  a 
later  modification  of  the  original  conception  of  Metschnikoff,  that 
the  invading  parasites  were  eaten  up  by  the  phagocytes,  and  was 
made  necessary  by  the  investigation  of  the  bactericidal  property  of 
the  body  juices.  The  experiments  of  Wright  and  Douglasf  indi- 
cate that  the  action  of  the  phagocytes  upon  the  bacteria  is  not 
immediate,  but  only  subsequent  to  a  preparative  action  upon  the 
organisms  by  substances  contained  in  serum,  to  which  they  have 
given  the  name  "Opsonins"  (Lat.  opsono,  "I  prepare  a  meal  for"). 
Long  before  Metschnikoff  began  his  studies  of  the  phagocytes 
Traube  and  GscheidelJ  observed  that  the  blood-plasma  possessed 
the  power  of  destroying  the  vitality  of  bacteria.  Grohman§  next 
observed  that  not  only  the  intravascular,  but  also  the  extra  vascular 
blood  possessed  this  property.  Further  studies  of  the  subject  were 
made  by  von  Fodor.||  The  systematic  investigation  of  the  bac- 
tericidal activity  of  blood-serum  in  -vitro  was  next  taken  up  by 
Fliigge,**  and  more  particularly  by  Nuttall,ft  who  found  that  dif- 

*  "Compte  rendu  de  1'Acad.  des  Sciences  de  Paris,"  1901,  cxxxm,  p.  244. 

f  "Proc.  Royal  Society  of  London,"  1904,  LXXXII,  p.  357. 

t  "  Jahresberichte  der  schles.  Ges.  f.  vaterl.  Kultur,"  1874. 

§  "  Untersuchungen  aus  dem  physiol.  Institut  zu  Dorpat,"  Dorpat,   1884; 
Kriiger. 

|j  "  Centralbl.  f.  Bakt.,"  etc.,  1890,  vn,  p.  753. 
**  "Zeitschrift  fur  Hygiene,"  Bd.  TV,  S.  208. 
tflbid.,  Bd.  iv,  353. 


io8  Immunity 

ferent  blood-serums  possessed  the  power  of  killing  bacteria  in 
large  numbers,  but  that  the  bactericidal  power  of  the  serum  soon 
disappeared,  after  which  the  serum  became  a  good  culture-medium 
for  the  very  bacteria  it  had  formerly  destroyed.  Metschnikoff 
objected  to  the  observations,  declaring  that  all  the  phenomena  were 
ultimately  referable  to  the  leukocytes,  so  Nuttall  investigated  peri- 
cardial  fluid  and  the  aqueous  humor  of  the  eye,  which  were  also 
found  to  possess  bactericidal  powers. 

The  matter  was  next  taken  up  by  Buchner  and  his  associates,* 
who  showed  that  the  blood-plasma  and  blood-serum  possessed 
exactly  the  same  bactericidal  effects  as  the  total  blood.  Buchner 
and  Nuttall  both  showed  that  the  exposure  of  the  bactericidal  fluids 
to  a  temperature  of  56°C.  for  a  few  hours  entirely  destroyed  their 
activity,  though  low  temperatures  were  without  effect  upon  them. 
Buchner  found  that  the  exposure  of  the  serum  to  sunlight  and  oxygen 
also  destroyed  the  bactericidal  power.  Neutralization  of  alkaline 
serum  did  not  destroy  its  activity,  but  when  the  serum  was  dialyzed 
and  the  NaCl  removed  from  it,  the  germicidal  power  was  lost,  to 
return  again  when  it  was  restored.  Buchner  called  the  bactericidal 
principle  alexin. 

Many  interesting  facts  were  collected  bearing  upon  the  bactericidal 
substance  or  alexin.  Thus  Morof  showed  that  it  was  proportionally 
more  active  in  sucking  infants  than  in  adults,  and  Ehrlich  and 
Brieger  J  found  that  it  passed  from  mother  to  offspring  in  the  milk. 

At  first  Buchner  regarded  alexin  as  an  albumin,  but  later§  he 
came  to  look  upon  it  as  a  proteolytic  enzyme,  this  view  no  doubt 
resulting  from  an  endeavor  to  explain  the  relation  of  alexin  to  im- 
munity against  intoxication,  in  which  it  was  necessary  to  show  that 
alexin  not  only  killed  bacteria,  but  also  destroyed  toxins. 

Hankin||  endeavored  to  show  that  there  were  differences  between 
the  substances  destroying  the  bacteria  and  those  acting  upon  their 
toxic  products.  To  the  whole  group  he  applied  the  term  defensive 
proteins.  Those  present  in  natural  immunity  he  called  sozins. 
those  found  in  acquired  immunity  phylaxins.  Sozins  with  bacteri- 
cidal activity  he  further  described  as  mycosozins,  those  with  toxin- 
destroying  activities  as  toxosozins.  Phylaxins  with  bactericidal 
action  were  called  my co phylaxins;  those  with  toxin-destroying 
properties  toxo phylaxins. 

Metschnikoff  found  it  unnecessary  to  modify  his  ideas,  but  per- 
sisted in  referring  all  the  phenomena  to  the  phagocytes  or  to  enzymes 
derived  from  them. 

At  this  point  it  will  be  evident  to  the  reader  that  the  phagocytic 

*"Centralbl.  f.  Bakt.,"  etc.,  1889,  Bd.  v,  817;  vi,  i;  "Archiv  fur  Hygiene," 
1891,  x,  S.  727;  "Centralbl.  f.  Bakt.,"  etc.,  1890,  vn,  76.    • 
f  "  Jahresb.  f.  Kinderheilkunde,"  v,  396. 
j  "Zeitschrift  fur  Hyg.,"  1893,  xni,  336. 
§  "Munch,  med.  Woch.,"  1899. 
If  "Centralbl.  f.  Bakt.,"  etc.,  xn,  Nos.  22,  23;  xiv,  No.  25. 


Defensive  Proteins,  etc.  109 

theory  and  the  humoral  theory  contain  indubitable  evidence  that 
both  the  body  cells  and  humors  are  important  factors  in  defending 
the  body  against  invading  organisms,  and  that  in  each  we  see  mechan- 
isms operative  in  certain  cases.  But  we  have  seen  that  both 
Metschnikoff  and  Buchner  are  obliged  to  strain  a  point  in  order  to 
meet  the  requirements  of  increasing  knowledge  of  the  subject  of 
immunity. 

Thus,  when  we  come  to  analyze  Buchner's  theory  of  alexins,  we 
find  that  if  natural  immunity  depends  upon  the  ability  of  the 
alexins  to  destroy  bacteria,  that  which  takes  place  in  vitro  should 
correspond  with  that  which  takes  place  in  vivo,  and  that  the  invasion 
of  the  animal's  body  by  bacteria  should  be  accompanied  by  diminu- 
tion of  the  bactericidal  substance  in  its  blood,  which  should  be  used 
up  before  the  bacteria  can  be  successful  in  their  invasion.  Experi- 
mental evidence  is,  however,  at  hand  to  show  that  this  is  not  always 
true. 

Behring  and  Nissen*  found  that  there  was  a  definite  relation  be- 
tween the  bactericidal  power  of  the  blood  in  vitro  and  the  resisting 
powers  of  a  large  number  of  animals  studied,  but  Lubarschf  showed 
the  remarkable  exceptions  of  the  rabbit,  which  is  highly  susceptible 
to  anthrax,  though  its  blood  is  highly  bactericidal  to  the  anthrax 
bacillus,  and  the  dog,  which  is  scarcely  susceptible  to  anthrax, 
though  its  blood  is  scarcely  bactericidal  to  the  bacillus. 

FluggeJ  found  the  bactericidal  power  of  the  blood  greatly  lessened 
in  thirty-six  hours  after  anthrax  infection,  and  Nissen  that  a  definite 
number  of  bacteria  could  be  killed  by  a  bactericidal  serum,  after 
which  the  alexin  became  inactive.  The  diminution  of  the  bac- 
tericidal power  was  shown  to  occur  both  in  the  animal  and  in  the 
test-tube.  He  also  showed  that  the  reactions  of  the  bactericidal 
serums  were  specific,  and  that  when  a  culture  of  one  kind  of  bacteria 
was  injected  into  an  animal,  the  immediate  effect  was  to  diminish 
the  activity  of  the  serum  for  that  species,  though  not  necessarily 
for  other  species.  The  diminution  of  bactericidal  energy  was  shown 
by  him  to  depend  upon  the  presence  of  the  bacteria,  as  the  injection 
of  filtrates  of  bacterial  cultures  did  not  affect  the  bactericidal 
properties  of  the  serum.  This  was  a  very  important  observation. 

There  is  a  correspondence  between  the  behavior  of  the  phagocytes 
and  the  body  juices.  When  the  activity  of  the  phagocytes  toward 
the  bacteria  is  increased,  the  bactericidal  activity  of  the  serum  is 
usually  intensified.  But  immunity  is  only  partly  explained  by 
alexins  and  bacteriolysis,  for  it  embraces  the  ability  of  the  organ- 
ism to  endure  the  effects  of  toxins  some  of  which  are  in  no  way 
connected  with  bacteria. 

Tolerance  to  certain  toxins  is,  of  course,  natural  to  many  animals, 

*  "Zeitschrift  fur  Hygiene,"  1890,  vm,  412. 
t  "Centralbl.  f.  Bakt.,"  etc.,  1889,  vi,  481. 
J  "Zeitschrift  fur  Hygiene,"  iv,2o8. 


no  Immunity 

and  tolerance  to  usually  destructive  toxins  natural  to  a  few.  This 
toxin-neutralizing  or  annulling  factor  cannot  be  identical  with  the 
bacteria-destroying  mechanism.  Cobbett,*  Roux  and  Mar  tin,  f 
and  BoltonJ  have  shown  that  horses  that  cannot  be  supposed  ever 
to  have  come  into  contact  with  diphtheria  bacilli,  vary  considerably 
in  their  resistance  to  diphtheria  toxin,  and  that  the  serum  of  the 
resisting  horses  contains  something  that  destroys  or  neutralizes  the 
toxin  in  vitro,  as  well  as  exerts  a  protective  influence  upon  animals 
into  which  it  is  injected.  This  substance  exerts  no  inimical  action 
upon  the  diphtheria  bacilli,  beyond  what  a  normal  serum  would  do, 
therefore  cannot  be  alexin,  but  must  be  antitoxin.  Abel§  found  that 
the  blood  of  healthy  men  occasionally  contained  some  substance 
capable  of  neutralizing  diphtheria  toxin;  Stern  found  one  normal 
serum  capable  of  protecting  against  typhoid  infection  and  Met- 
schnikoff  one  that  protected  against  cholera  infection.  Fischel 
and  Wunschheim||  found  newly  born  babies  immune  against  diph- 
theria, presumably  because  of  the  presence  of  a  small  quantity  of 
demonstrable  protective  substance  in  the  blood.  These  are, 
however,  peculiar  and  exceptional  cases. 

The  most  suggestive  and  fascinating  theory  of  immunity  is  that 
of  Ehrlich,  and  is  known  as  the  "  Seitenkettentheorie "  or  the 
"Lateral-Chain  Theory."** 

He  began  his  studies  by  an  investigation  into  the  nature  of  toxins 
and  their  mode  of  action.  The  discovery  that  there  was  no  con- 
stant relation  between  the  intoxicating  and  antitoxin  combining 
powers  of  diphtheria  toxic  bouillon  led  him  to  the  conclusion  that 
the  toxin  molecules  possessed  two  different  affinities,  which  he  de- 
scribed as  haptophorous  or  combining,  and  toxophorous  or  poisoning. 
The  former  were  constant,  the  latter  variable.  The  deterioration 
in  the  strength  of  the  toxic  filtrates  of  bouillon  cultures  of  diph- 
theria bacilli  was  shown  to  depend  upon  the  transformation  of  the 

*  "Lancet,"  Aug.  5,  1899,  n,  p.  532. 

t  "Ann.  de  1'Inst.  Pasteur,"  1894,  vin,  p.  615. 

j  "Jour,  of  Experimental  Medicine,"  July,  1896,  i,  No.  5. 

§  "Centralbl.  f.  Bakt.,"  etc.,  1895,  xvn,  p.  36. 

j|  "Zeitschr.  fur  Heilkunde,"  1895,  xvi,  p.  429-482. 

**  The  writings  of  Ehrlich  and  his  associates  are  so  numerous  and  scattered, 
and  often  so  fragmentary,  that  instead  of  referring  to  the  literature  according  to 
the  method  adopted  in  other  parts  of  this  work,  the  reader  who  desires  to  consult 
the  original  articles  can  best  do  so  by  making  use  of  the  following:  Ehrlich, 
"Die  Werthbemessung  des  Diphtheric  Heilserums,"  Klinisches  Jahrbuch, 
1897;  Ehrlich,  "Die  Konstitution  des  Diphtheriegiftes,"  Deutsche  med.  Woch., 
1898;  "Gesammelte  Arbeiten  zur  Immunitatsforschung,"  August  Hirschwald, 
Berlin,  1904— this  work  contains  the  collected  papers  of  Ehrlich  and  his  associates; 
Aschoff,  "Ehrlich's  Seitenkettentheorie  und  ihre  Anwendung  auf  die  Kunst- 
lichen  Immunusirungs-prozesse,"  Jena,  1902,  and  the  chapter  upon  "Wirkung 
und  Entstehung  der  Aktiven  Stoffe  im  Serum  noch  der  Seitenkettentheorie," 
by  Ehrlich  and  Morgenroth  in  Kolle  and  Wassermann's  "Handbuch  der  Patho- 
gene  Mikroorganismen,"  Jena,  1904,  Gustav  Fischer.  Readers  unacquainted 
with  the  German  language  may  find  the  essential  facts  in  Ehrlich's  Croonian 
Lecture,  Proceedings  of  the  Royal  Society  of  London,  1900,  LXVI,  p.  424,  and  in 
Welch's  "Huxley  Lecture,"  Medical  News,  1902,  LXXXI,  2,  p.  721. 


The  "Lateral-chain  Theory"  of  Immunity  in 

toxin  into  toxoids  which  were  not  poisonous,  and  was  shown  to  be 
quite  independent  of  the  antitoxin  combining  affinity  of  the  nitrate 
which  remained  unaltered.  The  inevitable  interpretation  seemed 
to  be  the  existence  in  the  bouillon  of  the  haptophorous  and  toxo- 
phorous  groups  described.  Similar  toxophorous  and  haptophorous 
groups  were  shown  to  exist  in  other  toxins — tetanolysin  by  Madsen, 
venoms  by  Myers,  and  milk-curdling  ferments  by  Morgenroth. 
The  neutralizing  action  of  the  antibodies  produced  in  the  blood  of 
animals  immunized  to  these  various  substances  depends  upon  the 
immediate  and  direct  combination  or  union  of  haptophorous  groups 
in  the  antibodies  with  corresponding  haptophorous  groups  of  the 
respective  toxins  or  active  bodies. 

The  physiological  activities  of  toxins  differ  from  those  of  alkaloids 
and  other  poisons  in  three  fundamentals:  first,  in  their  ability 
to  produce  antibodies  in  the  bodies  of  animals  into  which  they  are 
injected;  second,  the  manifestation  of  poisonous  action  only  after 
a  definite  incubation  period,  and  third  an  extremely  labile  com- 
position, by  which  the  toxin  becomes  quickly  transformed  to 
toxoids. 

ttsptophile  Toxof)hile         H&btofrhore        Toxofrhore 
group.        group.  group.  group., 

It  '  * 


Toxtn- 

Fig.  1 8. — Diagram  to  represent  the  combining  groups  of  the  cell  and  of  the 
toxin  respectively  (after  Ehrlich)  (Hewlett). 

Study  of  the  physiological  action  of  toxins  upon  the  cells  resulted 
in  showing  that  certain  definite  specific  affinities  existed,  and  that 
the  union  of  the  toxin  with  the  cell  antedated  the  production  of 
symptoms.  In  some  cases  it  was  even  found  possible  to  disconnect 
the  anchored  toxin  by  bringing  to  the  cells  haptophorous  groups  for 
which  the  haptophorous  elements  of  the  toxin  molecule  were  known 
to  have  an  active  affinity.  Donitz  determined  the  quantity  of 
tetanus  antitoxin  which,  injected  into  the  circulating  blood  imme- 
diately after  the  toxin,  absolutely  neutralized  it  and  rendered  all 
of  the  circulating  toxin  innocuous.  If  the  same  quantity  of  antitoxin 
was  given  seven  or  eight  minutes  after  the  injection  of  the  toxin, 
death  occurred  from  tetanus,  exactly  as  if  no  antitoxin  had  been 
given.  Evidently  the  toxin  had  anchored  itself  to  the  nerve-cells 
too  quickly  for  the  antitoxin  to  reach  and  combine  with  it.  Hey- 
mans  found  that  if  an  animal  was  injected  with  tetanus  toxin  and 
its  entire  blood  withdrawn  immediately  afterward  and  replaced  by 


ii2  Immunity 

transfusion,  it  died  of  typical  tetanus  because  in  the  brief  interval 
between  the  toxin  injection  and  the  transfusion,  the  toxin  molecules 
became  anchored  to  the  cell. 

The  ability  of  the  cells  thus  to  anchor  the  toxin  is  supposed  by 
Ehrlich  to  depend  upon  the  existence  of  haptophorous  combining 
affinities,  which  he  describes  as  receptors.  He  views  the  mode  of 
toxin  reception  as  depending  upon  a  mechanism  either  identical 
with  or  analogous  to  that  by  which  cellular  nutrition  is  maintained, 
and  points  out  that  in  the  case  of  methylene-blue  and  other  colored 
substances,  which  afford  an  opportunity  to  make  ocular  observations 
upon  the  absorption  of  the  pigment  by  the  cells,  only  certain  cells 
absorb  the  colors. 

Cell  nutrition  is  therefore  probably  carried  on  through  the  agency 
of  receptors  by  which  appropriate  nutrient  haptophorous  groups  are 
apprehended  and  utilized. 

The  following  somewhat  lengthy  quotation  from  his  "Croonian 
Lecture  upon  the  Lateral  Chain  Theory  of  Immunity,"  delivered 
before  the  Royal  Society  of  London,  March  22,  1900,  explains  the 
theory  in  Ehrlich's  own  words: 

"We  now  come  to  the  important  question  of  the  significance  of  the  toxophile 
groups  in  organs.  That  these  are  in  function  especially  designed  to  seize  on 
toxins  cannot  be  for  one  moment  entertained.  It  would  not  be  reasonable  to 
suppose  that  there  were  present  in  the  organism  many  hundreds  of  atomic  groups 
destined  to  unite  with  toxins,  when  the  latter  appeared,  but  in  function  really 
playing  no  part  in  the  processes  of  normal  life,  and  only  arbitrarily  brought  into 
relation  with  them  by  the  will  of  the  investigator.  It  would,  indeed,  be  highly 
superfluous,  for  example,  for  all  our  native  animals  to  possess  in  their  tissues 
atomic  groups  deliberately  adapted  to  unite  with  abrin,  ricin,  and  crotin,  sub- 
stances coming  from  far-distant  tropics." 

"One  may,  therefore,  rightly  assume  that  these  toxophile  protoplasmic  groups 
in  reality  serve  normal  functions  in  the  animal  organism,  and  that  they  only 
incidentally  and  by  pure  chance  possess  the  capacity  to  anchor  themselves  to 
this  or  that  toxin." 

"The  first  thought  suggested  by  this  assumption  was  that  the  atom  group 
referred  to  must  be  concerned  in  tissue  change;  and  it  may  be  well  here  to  sketch 
roughly  the  laws  of  cell  metabolism.  Here  we  must,  in  the  first  place,  draw  a 
clear  line  of  distinction  between  those  substances  which  are  able  to  enter  into 
the  composition  of  the  protoplasm,  and  so  are  really  assimilated,  and  those 
which  have  no  such  capacity.  To  the  first  class  belong  a  portion  of  the  food- 
stuffs, par  excellence;  to  the  second  almost  all  our  pharmacological  agents, 
alkaloids,  antipyretics,  antiseptics,  etc." 

"How  is  it  possible  to  determine  whether  any  given  substance  will  be  assimi- 
lated in  the  body  or  not?  There  can  be  no  doubt  that  assimilation  is  in  a  special 
sense  a  synthetic  process — that  is  to  say,  the  molecule  of  the  food-stuff  concerned 
enters  into  combination  with  the  protoplasm  by  a  process  of  condensation  in- 
volving loss  of  a  portion  of  its  water.  To  take  the  example  of  sugar,  in  the  union 
with  protoplasm,  not  sugar  itself  as  such,  but  a  portion  of  it,  comes  into  play, 
the  sugar  losing  in  the  union  some  of  its  characteristic  reactions.  The  sugar 
behaves  here  as  it  does,  e.g.,  in  the  glucosids,  from  which  it  can  only  be  obtained 
through  the  agency  of  actual  chemical  cleavage.  The  glucosid  shows  no  traces 
of  sugar  when  extracted  in  indifferent  solvents.  In  a  quite  analogous  manner  the 
sugar  entering  into  the  composition  of  albuminous  bodies  (glycoproteids)  cannot 
be  obtained  by  any  method  of  extraction,  at  least  not  until  chemical  composition 
has  previously  taken  place.  It  is,  therefore,  generally  easy  by  means  of  extrac- 
tion experiments  to  decide  whether  any  given  combination  in  which  the  cells 
take  part  Is,  or  is  not,  a  synthetic  one.  If  alkaloids,  aromatic  amines,  anti- 
pyretics, or  anilin  dyes  be  introduced  into  the  animal  body,  it  is  an  easy  matter, 


The  "Lateral-chain  Theory"  of  Immunity  113 

by  means  of  water,  alcohol,  or  acetone,  according  to  the  nature  of  the  body,  to 
remove  all  these  substances  quickly  and  easily  from  the  tissues." 

"This  is  most  simply  and  convincingly  demonstrated  in  the  case  of  the  anilin 
dyes.  The  nervous  system  stained  with  methylene-blue  or  the  granules 
of  the  cells  stained  with  neutral  red  at  once  yield  up  the  dye  in  the  presence  of 
alcohol.  We  are,  therefore,  obliged  to  conclude  that  none  of  the  foreign  bodies 
just  mentioned  enter  synthetically  into  the  cell  complex,  but  are  merely  con- 
tained in  the  cells  in  their  free  state."  ....  " Hence  with  regard  to  the 
pharmacologically  active  bodies  in  general,  it  is  not  allowable  to  assume  that 
they  possess  definite  atom  groups,  which  enter  into  combination  with  correspond- 
ing groups  of  the  protoplasm.  This  corresponds,  as  I  may  remark  beforehand, 
with  the  incapacity  of  all  these  substances  to  produce  antitoxins  in  the  animal 
body.  We  must,  therefore,  conclude  that  only  certain  substances,  food-stuffs, 
par  excellence,  are  endowed  with  properties  admitting  of  their  being,  in  the 
previously  defined  sense,  chemically  bound  by  the  cells  of  the  organism.  We 
are  obliged  to  adopt  the  view  that  the  protoplasm  is  equipped  with  certain  atomic 
groups,  whose  function  especially  consists  in  fixing  to  themselves  certain  food- 
stuffs of  importance  to  the  cell-life."  We  may  assume  that  the  protoplasm 
consists  of  a  special  executive  center,  in  connection  with  which  are  nutritive 
side-chains,  which  possess  a  certain  degree  of  independence  and  which  may 
differ  from  one  another  according  to  the  requirements  of  the  different  cells. 
And  as  these  side-chains  have  the  office  of  attaching  to  themselves  certain 
food-stifffs,  we  must  also  assume  an  atom-grouping  in  these  food-stuffs  them- 
selves, every  group  uniting  with  a  corresponding  combining  group  of  a  side-chain. 


Fig.  19. — Shows  how  the  haptophores  having  united,  the  toxophores  find  a  sec- 
ondary adaptation  to  the  cell,  and  so  can  poison  it  (after  Ehrlich)  (Hewlett). 

The  relationship  of  the  corresponding  groups,  i.e.,  those  of  the  food-stuff  and 
those  of  the  cell,  must  be  specific.  They  must  be  adapted  to  one  another,  as, 
e.g.,  male  and  female  screw  (Pasteur),  or  as  lock  and  key  (E.  Fischer).  From 
this  point  of  view,  we  must  contemplate  the  relation  of  the  toxin  in  the  cell." 

"We  have  already  shown  that  the  toxins  possess  for  the  antitoxins  an  attaching 
haptophore  group,  which  accords  entirely  in  its  nature  with  the  conditions  we 
have  ascribed  to  the  relation  existing  between  the  food-stuffs  and  the  cell  side- 
chains.  And  the  relation  between  toxin  and  cell  ceases  to  be  shrouded  in  mystery 
if  we  adopt  the  view  that  the  haptophore  groups  of  the  toxins  are  molecular 
groups  fitted  to  unite  not  only  with  the  antitoxins,  but  also  with  the  side-chains 
of  the  cells,  and  that  it  is  by  their  agency  that  the  toxin  becomes  anchored  to 
the  cells." 

"We  do  not,  however,  require  to  suppose  that  the  side-chains,  which  fit  the 
haptophore  group  of  the  toxins,  that  is,  the  side-chains  which  are  toxophile, 
represent  something  having  no  function  in  the  normal  cell  economy.  On  the 
contrary,  there  is  sufficient  evidence  that  the  toxophile  side-chains  are  the  same 
as  those  which  have  to  do  with  the  taking  up  of  the  food-stuffs  by  the  protoplasm. 
The  toxins  are,  in  opposition  to  other  poisons,  of  extremely  complex  structure, 
standing  in  their  origin  and  chemical  constitution  in  very  close  relationship  to 
the  proteids  and  their  nearest  derivatives.  It  is,  therefore,  not  surprising  that 
they  possess  a  haptophore  group  corresponding  with  that  of  a  food-stuff.  Along- 
side of  the  binding  haptophore  group,  which  conditions  their  union  to  the 
protoplasm,  the  toxins  are  possessed  of  a  second  group,  which  in  regard  to  the 
cell  is  not  only  useless  but  actually  injurious.  And  we  remember  that  in  the 
case  of  the  diphtheria  toxin  there  was  reason  to  believe  that  there  existed  along- 


ii4  Immunity 

side  of  the  haptophore  group  another  and  absolutely  independent  toxophore 
group."  ....  "  As  has  been  said,  the  possession  of  a  toxophile  group  by  the 
cell  is  the  necessary  preliminary  and  cause  of  the  poisonous  action  of  the  toxin." 
.  .  .  .  "If  the  cells  of  these  organs  [organs  essential  to  life]  lack  side-chains 
fitted  to  unite  with  them,  the  toxophore  group  cannot  become  fixed  to  the  cell, 
which  therefore  suffers  no  injury,  i.e.,  the  organism  is  naturally  immune.  One  of 
the  most  important  forms  of  natural  immunity  is  based  upon  the  circumstance 
that  in  certain  animals  the  organs  essential  to  life  are  lacking  in  those  haptophore 
groups  which  seize  upon  definite  toxins.  If,  for  example,  the  ptomaine  occurring 
in  sausages,  which  for  man,  monkeys,  and  rabbits  is  toxic  in  excessively  minute 
doses,  is  for  the  dog  harmless  in  quite  large  quantities,  this  is  because  the  binding 
haptophore  groups  being  wanting,  the  ptomaine  cannot,  in  the  dog,  enter  into 
direct  relation  with  organs  essential  to  life."  ....  "The  haptophore  group 
exercises  its  activity  immediately  after  injection  into  the  organism,  while  in  all 
toxins — with  the  perhaps  solitary  exception  of  snake-venom — the  toxophore 
group  comes  into  activity  after  the  lapse  of  a  longer  or  shorter  incubation  period 
which  may,  e.g.,  in  the  case  of  diphtheria  toxin,  extend  to  several  weeks." 

"The  theory  above  developed  allows  of  an  easy  and  natural  explanation  of 
the  origin  of  antitoxins.  In  keeping  with  what  has  already  been  said,  the  first 
stage  in  the  toxin  action  must  be  regarded  as  the  union  of  the  toxin  by  means  of 
its  haptophore  group  to  certain  'side-chains'  of  the  cell  protoplasm.  This 
union  is,  as  animal  experiments  with  a  great  number  of  toxins  show,  a  firm  and 
enduring  one.  The  side-chain  involved,  so  long  as  the  union  lasts,  cannot 


<£) 


Fig.  20. — Cells  with  various  receptors  or  haptophorous  groups  of  the  first 
order  (a),  adapted  to  combination  with  the  haptophorous  groups  (6)  of  various 
chemical  compounds  brought  to  them.  It  will  be  noted  that  there  is  no  mechan- 
ism by  which  the  toxophorous  elements  of  the  molecules  (c)  can  be  brought  to 
the  cell. 

exercise  its  normal  nutritive  physiological  function — the  taking  up  of  food-stuffs. 
It  is,  as  it  were,  shut  out  from  participating,  in  the  physiological  sense,  in  the 
life  of  the  cell.  We  are,  therefore,  now  concerned  with  a  defect  which,  according 
to  the  principles  so  ably  worked  out  by  Professor  Carl  Weigert,  is  repaired  by 
regeneration.  These  principles,  in  fact,  constitute  the  leading  conception  of  my 
theory.  If  after  union  has  taken  place  new  quantities  of  toxin  are  administered 
at  suitable  intervals  and  in  suitable  quantities,  the  side-chains,  which  have  been 
reproduced  by  the  regenerative  process,  are  taken  up  anew  into  union  with  the 
toxin,  and  so  again  the  process  of  regeneration  gives  rise  to  the  formation  of 
fresh  side-chains.  In  the  course  of  the  progress  of  typical  systematic  immuni- 
zation, as  this  is  practised  in  the  case  of  diphtheria  and  tetanus  toxin  especially, 
the  cells  become,  so  to  say,  educated  or  trained  to  reproduce  the  necessary  side- 
chains  in  ever-increasing  quantity.  As  Weigert  has  confirmed  by  many  ex- 
amples, this,  however,  does  not  take  place  by  the  simple  replacement  of  the  defect; 
the  compensation  proceeds  far  beyond  the  necessary  limit;  indeed,  overcom- 
pensation  is  the  rule.  Thus  the  lasting  and  ever-increasing  regeneration  must 
finally  reach  a  stage  at  which  such  an  excess  of  side-chains  is  produced  that,  to 
use  a  trivial  expression,  the  side-chains  are  present  in  too  great  a  quantity  for  the 
cell  to  carry  and  are,  after  the  manner  of  a  secretion,  handed  over  as  needless 
ballast  to  the  blood.  Regarded  in  accordance  with  this  conception,  the  anti- 
toxins represent  nothing  more  than  side-chains  reproduced  in  excess  during  re- 
generation'and  therefore  pushed  of  from  the  protoplasm  and  so  coming  to  exist  in 
the  free  state." 


The  "Lateral-chain  Theory"  of  Immunity 


"In  the  first  place,  our  theory  affords  an  explanation  of  the  specific  nature  of 
the  antitoxins,  that  tetanus  antitoxin  is  only  caused  to  be  produced  by  tetanus 
toxin,  and  diphtheria  antitoxin  through  diphtheria  toxin.  This  very  specific 
nature  of  the  affinity  between  toxin  and  cell  is  the  necessary  preliminary  and 
cause  of  the  toxicity  itself.  Further,  our  theory  makes  it  easy  to  understand  the 
long-lasting  character  of  the  immunity  produced  by  one  or  several  administra- 
tions of  toxin,  and  also  the  fact  that  the  organism  reacts  to  relatively  small 


Figs.  21  and  22. — Show  the  regeneration  of  the  cell-haptophores  or  receptors  to 
compensate  for  the  loss  of  those  thrown  out  of  service. 

quantities  of  toxin  by  the  production  of  very  much  greater  quantities  of  anti- 
toxin. By  the  act  of  immunization,  certain  cells  of  the  organism  become  con- 
verted into  cells  secreting  antitoxin  at  the  same  rate  as  this  is  excreted.  New 
quantities  of  antitoxin  are  constantly  produced,  and  so  throughout  a  long  period 
the  antitoxin  content  of  the  serum  remains  nearly  constant.  The  secretory 
nature  of  the  formation  of  antitoxins  has  been  very  strikingly  illustrated  by  the 
beautiful  experiments  of  Salmonson  and  Madsen,  who  have  shown  that  pilo- 


Fig.  23. — Shows  the  number  of 
haptophores  regenerated  by  the  cell 
becoming  excessive;  they  are  thrown 
off  into  the  tissue  juice. 


V 

it! 


Fig.  24. — Explains  what  antitoxins 
are  and  how  they  are  formed.  The 
liberated  receptors  in  the  tissue  juice 
and  in  the  blood,  possess  identical  com- 
bining affinities  with  those  upon  the 
cell,  and  meeting  the  adapted  hapto- 
phorous  elements  in  the  blood,  com- 
bine with  them,  thus  keeping  them 
from  the  cells. 


carpine,  which  augments  the  secretion  of  most  glands,  also  occasions  in  immu- 
nized animals  a  rapid  increase  in  the  antitoxin  content  of  the  serum." 

"The  production  of  antitoxins  must,  in  keeping  with  our  theory,  be  regarded 
as  a  function  of  the  haptophore  group  of  the  toxin,  and  it  is  easy  therefore  to 
understand  why,  out  of  the  great  number  of  alkaloids,  none  are  in  a  position  to 
cause  the  production  of  antitoxins.  Conversely,  indeed,  I  recognize  in  this 
incapacity  of  the  alkaloids,  in  opposition  to  the  toxins,  to  produce  antitoxins 
a  further  and  salient  proof  of  the  truth  of  the  deduction  I  have  previously  based 
on  chemical  grounds,  that  the  alkaloids  possess  no  haptophore  group  which 


n6  Immunity 

anchors  them  to  the  cells  of  organs.  To  formulate  a  general  statement,  the 
capacity  of  a  body  to  cause  the  production  of  antitoxin  stands  in  inseparable 
connection  with  the  presence  of  a  haptophore  atomic  group.  In  the  formation 
of  antitoxin  the  toxophore  group  of  the  toxin  molecule  is,  on  the  contrary,  of 
absolutely  no  moment.  But  the  toxoid  modification  of  the  toxins,  in  which  the 
haptophore  group  of  the  toxin  is  retained,  while  the  toxophore  group  has  ceased 
to  be  active,  possesses  the  property  of  producing  antitoxins.  Indeed,  in  some 
cases  of  extremely  susceptible  animals,  immunity  can  only  be  attained  by  means 
of  the  toxoids,  and  not  by  the  too  strongly  acting  toxins."  ....  "The 
symptoms  of  illness  due  to  the  action  of  the  toxophore  group,  therefore,  play  no 
part  in  the  production  of  antitoxin."  The  effect  of  enzymes  upon  the  organism 
with  the  production  of  antibodies,  and  the  "specific  precipitins"  caused  by  the 
injection  of  milk,  albumin,  and  peptones  into  animals  may  be  looked  upon  as 
"having  their  origin  in  the  most  widely  diverse  organs,  and  representing  nothing 
more  than  nutritive  side-chains,  which  in  the  course  of  the  normal  nutritive 
processes  have  been  developed  in  excess  and  pushed  off  into  the  blood." 

"Much  more  complex  than  in  the  cases  hitherto  discussed  are  the  conditions 
when,  instead  of  the  relatively  simple  metabolic  products  of  microbes,  the  living 
micro-organisms  themselves  come  to  be  considered,  as  in  immunization  against 
cholera,  typhoid,  anthrax,  swine-fever,  and  many  other  infectious  diseases. 
Thus  there  come  into  existence,  alongside  of  the  antitoxins  produced  as  a  result 
of  the  action  of  the  toxins,  manifold  other  reaction  products.  This  is  because 
the  bacterium  is  a  highly  complicated  living  cell  of  which  the  solution  in  the 
organism  yields  a  great  number  of  bodies  of  different  nature,  in  consequence  of 
which  a  multitude  of  'antikorper'  are  called  into  existence.  Thus  we  see,  as  a 
result  of  the  injection  of  bacterial  cultures,  that  there  arise  alongside  of  the 
specific  bacteriolysins,  which  dissolve  the  bacteria,  other  products,  as,  for  example, 
the  'coagulins'  (Kraus,  Bordet),  i.e.,  substances  which  are  able  to  cause  the 
precipitation  of  certain  albuminous  bodies  contained  in  the  culture  fluid  injected; 
also  the  much-discussed  agglutinins  (Durham,  Gruber,  Pfeiffer),  the  antifer- 
ments  (von  Diingern),  and  no  doubt  many  other  bodies  which  have  not  yet  been 
recognized.  It  is  by  no  means  unlikely  that  each  of  these  reaction  products 
finds  its  origin  in  special  cells  of  the  body;  on  the  other  hand,  it  is  quite  likely 
that  the  formation  of  any  single  one  of  these  bodies  is  not  of  itself  sufficient  to 
confer  immunity.  Thus,  in  the  case  of  the  introduction  of  bacteria  into  the 
body  we  have  to  do  with  a  many-sided  production  of  different  forms  of  'anti- 
korper,' .each  of  which  is  directed  only  against  one  definite  quality  or  metabolic 
product  of  the  bacterial  cell.  Accordingly,  in  recent  times,  the  practice  of  using 
for  the  production  of  immunization  definite  toxic  bodies  isolated  from  the 
bacterial  cells  has  been  more  and  more  given  up,  and  for  this  purpose  it  is  now 
regarded  as  important  to  employ  the  bacterial  cells  as  intact  as  possible."  .  .  . 
"The  most  interesting  and  important  substances  arising  during  such  an  immuniz- 
ing process  are  without  doubt  the  bacteriolysins."  ....  "Belfanti  and  Car- 
bone  first  discovered  the  remarkable  fact  that  horses  which  had  been  treated 
with  the  blood-corpuscles  of  rabbits  contain  in  their  serum  constituents  which 
are  poisonous  for  the  rabbit,  and  for  the  rabbit  only."  .  .  .  "Bordet  showed 
shortly  thereafter  that  in  the  case  quoted,  there  was  present  in  the  serum  a 
specific  hemolysin  which  dissolved  the  corpuscles  of  the  rabbit.  He  also  proved 
that  these  hemolysins — as  had  already  been  shown  by  Buchner  and  Daremberg 
in  the  case  of  similarly  acting  bodies  which  are  present  in  normal  blood — lost 
their,  sol  vent  property  on  being  maintained  during  half  an  hour  at  a  temperature 
of  SS°C.  Bordet  added,  further,  a  new  fact,  that  the  blood-solvent  property 
of  those  sera  which  had  been  deprived  of  solvent  power  by  heat,  the  solvent 
action  could  be  restored  if  certain  normal  sera  were  added  to  them.  By  this 
important  observation  an  exact  analogy  was  established  with  the  facts  of 
bacteriolysis  as  elicited  by  the  work  of  Pfeiffer,  Metschnikoff,  and  Bordet." 

.  .  .  "In  collaboration  with  Dr.  Morgenroth,  I  have  sought  in  regard  to  this 
question,  for  which  hemolysis  offered  prospects  favorable  to  experimentation, 
to  make  clear  the  mechanism  concerned  in  the  action  of  these  two  compounds — 
the  stable,  which  may  be  designated  'immune  body,'  and  the  unstable,  which 
may  be  designated  'complement' — which  acting  together  effect  the  solution  of 
the  red  blood-corpuscles.  For  this  purpose,  in  the  first  place,  solutions  containing 
either  only  the  'immune  body'  or  only  the  'complement'  were  brought  in  contact 
with  suitable  blood-corpuscles,  and  after  separation  of  the  fluid  and  the  corpuscles 


The  " Lateral-chain  Theory"  of  Immunity  117 

by  centrifugalization,  we  investigated  whether  these  substances  had  been  taken 
up  by  the  red  corpuscles  or  remained  behind  in  the  fluid.  The  proof  of  its  loca- 
tion in  the  one  position  or  in  the  other  was  readily  forthcoming,  since  to  restore 
the  hemolysin  to  its  former  activity,  it  was  only  necessary  to  add  to  the  '  immune 
body'  a  fresh  supply  of  'complement,'  or  to  the  'complement'  a  fresh  supply  of 
'immune  body'  in  order  that  the  presence  of  the  hemolysin  in  its  integrity  might 
be  shown  by  the  occurrence  of  solution  of  the  red  cells.  The  experiments 
proved  that,  after  centrifugalizing,  the  'immune  body'  is  quantitatively  bound  to 
the  red  blood-corpuscles,  and  that  the  'complement,'  on  the  contrary,  remains 
entirely  behind  in  the  fluid.  The  presence  of  the  two  components  in  contact 
with  blood-corpuscles  only  occasions  the  solution  of  these  at  higher  temperatures, 
and  not  at  o°C.  And  an  active  hemolytic  serum  (with  'immune  body'  and 
'complement'  both  present)  having  been  placed  in  contact  with  red  blood- 
corpuscles  and  maintained  for  a  while  at  o°C.,  it  was  found  after  centrifugalizing 
that,  under  these  circumstances  also  the  'immune  body'  had  united  with  the 
red  blood-corpuscles,  but  that  the  'complement'  remained  in  the  serum.  This 
experiment  showed  that  both  components  must,  at  a  tem- 
perature of  o°C.,  have  existed  alongside  of  one  another  in 
a  free  condition."  .... 

"But  when  analogous  experiments  were  undertaken  at 
a  higher  temperature  it  was  found  that  both  components 
were  retained  in  the  sediment. 

"These  facts  can  only  be  explained  by  making  certain 
assumptions  regarding  the  constitution  of  the  two  compo- 
nents, i.e.,  of  the  'immune  body'  and  the 'complement.' 
In  the  first  place,  two  haptophore  groups  must  be  as- 
cribed to  the  *  immune  body,'  one  having  affinity  for  a 
corresponding  haptophore  group  of  the  red  blood-corpuscles 
and  with  which  at  a  lower  temperature  it  quickly  unites,  Fig.  25. — Com- 
and  another  haptophore  group  of  a  lesser  chemical  affinity,  bination  of  cell  (a), 
which  at  a  higher  temperature  becomes  united  with  the  amboceptor  (6),  and 
'complement'  present  in  the  serum.  Therefore  at  the  complement  (c). 
higher  temperature  the  red  blood-corpuscles  will  draw  to  The  amboceptor 
themselves  those  molecules  of  the  'immune body'  which  in  may  unite  with  the 
the  fluid  have  previously  become  united  to  the  'comple-  cell,  but  cannot  af- 
ment.'  In  this  case  the  'immune  body'  represents  in  a  feet  it  alone.  The 
measure  the  connecting  chain  which  binds  the  comple-  complement  cannot 
ment  to  the  red  blood-corpuscles  and  so  brings  them  unite  with  the  cell 
under  its  deleterious  influence.  Since  under  the  influence  except  through  the 
of  the  'complement' — at  least,  in  the  case  of  the  bacteria  amboceptor,  having 
— appearances  are  to  be  observed  (for  example,  in  the  no  adaptation  to  the 
Pf  eiffer  phenomenon)  which  must  be  regarded  as  analogous  cell  directly, 
to  digestion,  we  shall  not  seriously  err  if  we  ascribe  to 

this  'complement'  a  ferment-like  character."  ....  "Having  obtained  a 
precise  conception  of  the  method  of  action  of  the  lysins  of  the  serum — of  the 
hemolysins,  and  thereby  also  of  the  bacteriolysins — it  becomes  possible  for  us  to 
attempt  to  solve  the  mystery  of  the  origin  of  these  bodies.  I  have  in  the  begin- 
ning of  this  lecture  fully  developed  the  'side-chain  theory,'  according  to  which 
the  antitoxins  are  merely  certain  of  the  protoplasm  'side-chain'  which  have 
been  produced  in  excess  and  pushed  off  into  the  blood. 

"The  toxins  as  secretion  products  of  the  cells  are  in  all  likelihood  still  relatively 
uncomplicated  bodies;  at  least  by  comparison  with  the  primary  and  complex 
albumins  of  which  the  living  cell  is  composed. 

"If  we  now  recognize  that  the  different  lysins  arise  only  through  absorption 
of  highly  complex  cell  material — such  as  red  blood-corpuscles  or  bacteria — then 
the  explanation,  in  accordance  with  what  I  have  said,  is  that  there  are  present 
in  the  organism  'side-chains'  of  a  special  nature,  so  constituted  that  they  are 
endowed  not  only  with  an  atomic  group  by  virtue  of  the  affinities  of  which  they 
are  enabled  to  pick  up  material,  but  also  with  a  second  atomic  group,  which,  being 
ferment-loving  in  its  nature,  brings  about  the  digestion  of  the  material  taken  up. 
Should  the  pushing  off  of  these  'side-chains'  be  forced,  as  it  were,  by  immuni- 
zation, then  the  'side-chains'  thus  set  free  must  possess  both  groups,  and  will, 
therefore,  in  their  characteristics  entirely  correspond  with  what  we  have  placed 
beyond  doubt  as  regards  the  'immune  body'  of  the  hemolysin." 


n8  Immunity 

An  analysis  of  this  theory  shows  complete  natural  immunity 
to  depend  upon  the  absence  of  haptophore  groups  (receptors)  by 
which  the  toxins  can  be  united  to  the  cells.  Extreme  sensitivity 
or  susceptibility  probably  depends  upon  the  adapted  haptophores 
being  present  or  at  least  most  numerous  upon  the  cells  of  highly 
vital  organs;  comparative  insensitivity  or  insusceptibility  upon 
the  fact  that  the  greater  number  of  haptophore  groups  are  attached 
to  comparatively  unimportant  cells  whose  combining  affinities 
have  to  be  satisfied  before  combination  with  more  vital  cells  can 
be  accomplished.  In  some  cases  natural  immunity  is  increased  by 
the  presence  of  free  haptophore  groups  (antitoxin)  in  the  blood. 

Acquired  immunity  against  toxins  depends 
upon  the  regeneration  of  the  cellular  hapto- 
phores or  receptors  which,  being  liberated 
into  the  body  juices,  fix  the  haptophores  of 
the  toxin  molecules  before  they  are  able  to 
reach  the  cells  themselves.  Antitoxins  and 
other  anti-bodies,  including  the  lysins,  consist 
of  liberated  cellular  haptophores  or  receptors, 
the  former  having  a  single  combining  affinity, 
the  latter  a  double  combining  affinity,  by 
which  they  unite,  on  the  one  hand,  with  the 
cel1  to  be  dissolved,  on  the  other  with  the 
order  (a)  by  which  the  complement  by  which  it  is  to  be  dissolved, 
cells  fix  useful  molecules,  Antibodies  having  this  double  combining 
of  albumins,  etc.,  on  one  ~,  .,  ,  i_  n  j  «  *  »  i 

hand  (6),  and  zymogen    affinity  have  been  called  "amboceptors"  by 

molecules  (c)  on  the  other    Ehrlich.     They  are  variously  known  in  dif- 

hand,andmakeuseofthe    ferent  writings  as  " immune  bodies,"  ambo- 

one    substance    through 

the  action  of  the  other.      ceptors,  substance  sens^b^l^satr^ce,  desmon,  and 

fixateur.     The     "complement"     or     "addi- 

ment"  of  Ehrlich  is  also  called  alexin  and  cytase.  Ehrlich  con- 
ceives every  amboceptor  and  every  complement  to  be  specific,  but 
Bordet  and  others,  while  admitting  that  the  amboceptor  is  specific, 
hold  that  there  is  but  one  complement  or  cytase. 

It  has  already  been  said  that  MetschnikofFs  primitive  con- 
ception of  the  body  being  defended  against  infection  through  the 
phagocytic  incorporation  and  digestion  of  the  microparasites,  has 
had  to  be  modified  to  conform  to  the  increasing  information  upon 
the  immunity  reactions.  He  has  persistently  clung  to  the  idea  that 
the  phagocytes  are  the  essential  factors,  but  has  changed  the  con- 
ception of  " phagocytosis''  to  make  it  applicable  to  the  new  require- 
ments. He  now  teaches  that  when  invasive  micro-organisms  enter 
the  body,  chemotactic  influences  determine  that  they  shall  be  met 
by  phagocytes.  If  the  invading  micro-organisms  are  too  powerful 
and  the  phagocytes  are  killed,  phagolysis  or  dissolution  of  the  phago- 
cytes liberates  their  enzymes  into  the  blood.  These  liberated 
enzymes  still  act  deleteriously  upon  the  invaders,  tending  to  ag- 


The  "Lateral-chain  Theory"  of  Immunity  119 

glutinate — 'aggregate  them  in  clumps — and  sensitize  them  to  the 
future  action  of  other  phagocytes  by  which  they  may  be  taken 
up.  Through  extensive  phagolysis,  and  the  liberation  of  large 
quantities  of  the  enzyme  contents  of  the  phagocytes  into  the  blood, 
the  plasma  and  serum  acquire  a  "fixing"  or  "sensitizing"  quality 
from  the  macrocytase  of  the  macrophages,  which  is  the  "fixateur" 
or  "substance  sensibilisatrice"  and  a  bacteria-dissolving  quality 
forms  another  enzyme,  microcytase,  from  the  microphages.  Thus, 
we  find  that  Metschnikoff  is  prepared  to  account  for  the  "ambo- 
ceptor"  or  "immune  body"  of  Ehrlich,  which  is  the  macrocytase, 
and  the  "complement,"  which  is  the  "microcytase"  In  cases 
where  the  bacteria  exert  a  negatively  chemotactic  influence  upon 
the  leukocytes,  no  immunity  exists. 

The  antitoxins  are  similarly  accounted  for  by  Metschnikoff:  the 
cellular  digestive  enzymes  exert  their  action  not  only  upon  the 
microparasites,  but  also  upon  their  products,  fixing  or  otherwise 
altering  them  until  they  can  be  finally  destroyed. 

It  will  thus  be  seen  that  the  two  chief  theories  of  immunity,  though 
they  appear  discordant  when  explained  independently  of  one 
another,  can  be  fairly  well  harmonized.  Ehrlich  believes  the  im- 
mune bodies  to  be  the  products  of  those  cells  of  the  body  with  whose 
haptophile  combining  groups  the  haptophore  groups  of  the  antigen 
engaged,  and  does  not  attribute  the  function  to  any  particular 
group  of  cells;  Metschnikoff  attributes  all  the  activities  to  the 
phagocytes,  and  especially  the  leukocytes.  Ehrlich  looks  upon  the 
phenomena  as  chemical  and  pictures  them  as  taking  places  inde- 
pendently of  the  cells;  Metschnikoff  looks  upon  them  as  vital  and 
brought  about  by  the  agency  of  living  cells.  Both  theories  are 
ultimately  chemical. 

The  fundamental  ideas  embodied  in  the  ''lateral-chain  theory"  of  immunity 
may,  by  reversing  the  hypothesis  and  considering  the  bacterial  instead  of  the 
body  cells  to  be  upon  the  defensive,  be  made  to  explain  other  phenomena  of 
immunity.  Walker*  seems  to  have  been  the  pioneer  in  this  field,  and  his 
researches  show  that  it  is  possible  to  immunize  bacteria  against  "immune 
serums"  by  cultivating  them  in  media  containing  increasing  proportions  of  the 
immune  serums.  The  bacteria  thus  cultivated  were  of  increased  virulence. 
The  idea  was  further  amplified  by  Welch  in  his  Huxley  Lecture,  f  The  micro- 
organismal  cells  must  be  regarded  as  endowed  with  receptors  of  their  own,  fitted 
for  combination  with  adapted  haptophorous  elements  in  the  juices  reaching 
them,  and  therefore  capable  of  reacting  toward  such  substances  exactly  as  do  the 
cells  of  the  host.  As  the  host  reacts  toward  the  active  products  of  the  bacteria, 
so  the  bacteria  react  toward  the  defensive  products  of  the  host,  and  as  the  cells 
of  the  former  are  stimulated  to  the  production  of  immune  bodies  that  shall  facili- 
tate bacteriolysis,  so  the  latter  are  stimulated  to  antagonize  their  action  by 
producing  neutralizing  bodies.  These  neutralizing  bodies  by  which  the  defenses 
of  the  host  are  broken  down  are  among  those  described  by  BailJ  as  "aggressins." 

Thus,  as  the  cells  of  the  host  invaded  are  constantly  reacting  to  the  active 

*  "Jour,  of  Path,  and  Bact.,"  March,  1902,  vm,  No.  i,  p.  34. 

t  "British  Medical  Journal,"  Oct.  n,  1902,  p.  1105;  "Medical  News,"  Oct. 
18,  1902. 

}  "Wiener  klin.  Woch.,"  1905,  Nos.  9,  14,  16,  17;  "Berl.  klin.  Woch.,"  1905, 
No.  15;  "Zeitschr.  f.  Hyg.,"  1905,  Bd.  i,  No.  3. 


I2O  Immunity 

bodies  produced  by  the  invading  parasites,  so  the  latter  are  reacting  toward  the 
defensive  products  of  the  former.  If  the  reactive  processes  of  the  host  predomi- 
nate, immunity  and  the  destruction  of  the  parasites  result;  if  those  of  the  bacteria 
predominate,  increased  virulence,  facilitated  invasion,  and  death  of  the  host  may 
result.  This  hypothesis  also  serves  to  make  clear  why  micro-organisms  entering 
the  body  not  infrequently  show  a  marked  tendency  to  colonize  in  certain  organs 
and  tissues  in  preference  to  others. 

Supposing  accident  to  determine  the  tissue  in  which  the  primary  infection 
has  taken  place,  a  longer  or  shorter  residence  in  that  tissue,  with  the  resulting 
more  or  less  marked  acquired  immunity  against  the  defensive  activities  of  that 
tissue,  endow  the  organism  with  a  higher  degree  of  virulence  for  it  than  for  other 
tissues,  so  that  if  at  some  future  time  the  organism  entering  the  circulation  of  a 
new  host  were  able  to  colonize  in  any  tissue  of  the  body,  its  activities  could  be 
more  easily  and  more  successfully  manifested  in  that  to  which  it  had  already 
become  accustomed,  and  to  which  it  had  acquired  a  peculiar  adaptability.  This 
adaptability  has  been  made  the  subject  of  interesting  experimental  demonstra- 
tion by  Forssner*  in  his  work  upon  the  intravenous  injection  of  streptococci. 

SPECIAL  PHENOMENA  OF  INFECTION  AND  IMMUNITY 

Certain  phenomena  which  present  themselves  in  the  course 
of  infection  and  immunity,  to  which  reference  has  already  been 
casually  made,  must  now  be  considered  in  detail. 

SPECIFIC  PRECIPITATION 

Specific  precipitation  is  the  coagulation  or  precipitation  of  an  anti- 
gen by  its  specific  antibody.  In  1897  Krausf  while  studying  the 
"specific  reactions  produced  by  homologous  serums  with  germ- 
free  filtrates  of  bouillon  cultures,  of  cholera,  typhoid  and  plague 
bacteria,"  observed  that  immune  serum  brought  into  contact  with 
the  respective  culture  filtrate  occasioned  a  precipitate  specific  in 
nature,  to  which  he  gave  the  name  "  specific  precipitate.'! 

Bordetf  and  Tchistowitch§  showed  that  the  phenomenon  was 
of  wide  occurrence  and  had  a  broad  significance,  for  they  discovered 
that  when  the  serum  of  one  animal  was  injected  into  another  ani- 
mal of  different  kind,  some  reaction  took  place  in  the  injected  ani- 
mal, which  caused  a  precipitate  to  form  whenever  the  serums  of 
the  two  animals  were  being  subsequently  brought  together  in  a 
test-tube.  The  same  was  found  true  of  milk.  When  an  animal  was 
injected  with  the  milk  of  a  different  kind  of  animal,  its  serum  ac- 
quired the  property  of  causing  a  precipitate  to  form  when  its  serum 
and  filtered  milk  were  mixed  together  in  a  test-tube.  The  substance 
or  factor  inducing  the  precipitation  was  called  "precipitin"  or 
"coagulin."  Myers,  ]  Jacoby,**  Nolf,ft  and  others  showed  that 
the  faculty  of  provoking  specific  precipitins  was  common  to  many 
albuminous  bodies — 'albumen,  globulin,  albumose,  peptone,  ricin, 
etc.  Kraus  in  his  original  communication  dwelt  upon  the  specific 
nature  of  the  precipitation,  and  was  corroborated  by  Fish,|J  Wasser- 

*  "Nordiskt  Medicinskt  Archiv,"  1902,  Bd.  xxxv,  p.  i. 

t  "Wiener  klin.  Woch.,"  1897,  No.  32. 

J  "Ann.  de  ITnst.  Pasteur,"  1899,  p.  173. 

§  "Ann.  de  ITnst.  Pasteur,"  1899,  p.  406. 

|T"  Centralbl.  f .  Bakt.,"  etc.,  1900,  Bd.  xxxm,  and  "  The  Lancet,"  1900,  n,  p.  98. 
**"Arch~HT  fQr  exper  Path.  u.  Pharmak.,"  1900. 
ft  '*  Ann.  de  ITnst.  Pasteur,"  1900,  p.  297. 
it  "Courier  of  Medicine,"  St.  Louis,  Feb.,  1900. 


The  Specific  Precipitins 


121 


mann,*  Morgenroth,  and  others,  by  whom  it  has  been  shown  that 
the  reaction  is  sufficiently  accurate  to  make  possible  the  differentia- 
tion of  human  and  goat's  milk.  The  most  important  practical 
application  of  the  specific  character  of  the  precipitins,  however, 
came  through  Uhlenhuthf  and  Wassermann,f  who  made  use  of  it 
for  the  differentiation  of  bloods  for  forensic  purposes. 

Uhlenhuth  gave  rabbits  intraperitoneal  injections  of  10  cc.  of 
defibrinated  blood  at  intervals  of  from  six  to 
eight  days  and  found  the  blood-serum  strongly 
precipitant  after  the  fifth.  He  used  such  serum 
for  testing  the  reaction  with  the  bloods  of  oxen, 
horses,  donkeys,  pigs,  sheep,  dogs,  cats,  deer, 
hares,  guinea-pigs,  rats,  mice,  rabbits,  chickens, 
geese,  turkeys,  pigeons,  and  men. 

The  method  of  making  the  test  is  important, 
as  carelessness  of  detail  will  interfere  with  the 
accuracy  of  the  result.  The  blood  to  be  tested 
is  diluted  about  1:100,  or  until  it  has  a  feeble  red 
color,  with  tap  water,  and  then  freed  from  cor- 
puscular stroma  by  filtration  or  decantation. 
Two  cubic  centimeters  of  it  are  placed  in  a 
small  test-tube,  and  further  diluted  with  an 
equal  quantity  of  physiological  salt  solution  (if 
more  water  be  added  a  precipitate  of  globulin 
might  take  place  and  spoil  the  experiment). 
To  such  a  prepared  blood  solution,  from  six  to 
eight  drops  of  the  immune  serum  are  added. 
If  the  diluted  blood  come  from  the  same  kind  of 
animal  as  that  whose  blood  was  used  to  immunize 
the  animal  furnishing  the  test  serum,  immediate 
clouding  takes  place,  and  a  flocculent  precipi- 
tate forms.  The  precipitate  never  occurs  with 
any  other  blood. 

Wassermann  and  Schutze§  prepared  a  test 
serum  by  injecting  rabbits  with  human  blood, 
precipitating  powers  upon  twenty-three  other  kinds  of  blood  and 
found  no  precipitate  except  with  the  blood  of  a  baboon,  but  the  re- 
action in  that  case  was  not  nearly  so  marked  as  with  human  blood. 

The  most  interesting  and  one  of  the  most  important  biological 
applications  of  this  phenomenon  is  by  Nuttall,  whose  work,  "  Blood 
Immunity  and  Blood  Relationship"  (Cambridge,  1904),  should  be 
read  by  all  who  wish  to  study  the  subject  for  its  scientific  interest 
as  a  means  of  determining  the  blood  relationship  of  animals,  or  its 

*  "Verhandl.  d.  Kong.  f.  innere  Med.,"  1900,  501,  Wiesbaden. 
f  "Deutsche  med.  Woch.,"  1900  and  1901. 

i"Samml.  klin.  Vortr.  von  Volkman,"  Leipzig,  Verlag  von  Breitkopf  and 
Hartel,  1902. 

§  "Deutsche  med.  Wochenschrift,"  1900,  No.  30. 


Fig.  27. — Poly- 
ceptor  (Ehrlich  and 
Marshall)  such  as 
can  be  conceived  to 
occur  in  hemolysis 
and  bacteriolysis 
where  various  com- 
plements are  en- 
gaged, a,  Receptor 
of  bacterial  cell;  b, 
cytophil  group  of 
the  amboceptor;  c, 
dominating  comple- 
ment; d,  subordinate 
complement;  a,  /3, 
c  o  m  p  1  e  m  entophil 
groups  of  the  ambo- 
ceptor, a  for  the 
dominating,  /3  for 
the  subordinate 
complements. 

They  tested  its 


122  Immunity 

practical     medicolegal    importance    in    recognizing    blood-stains. 
Nuttall  comes  to  the  following  conclusions: 

"(i)  The  investigations  we  have  made  confirm  and  extend  the  observations 
of  others  with  regard  to  the  formation  of  specific  precipitins  in  the  blood-serum 
of  animals  treated  with  various  sera.  (2)  These  precipitins  are  specific,  although 
they  may  produce  a  slight  reaction  with  the  sera  of  allied  animals.  (3)  The  sub- 
stance in  serum  which  brings  about  the  formation  of  a  precipitin,  as  also  the  pre- 
cipitin  itself,  are  remarkably  stable  bodies.  (4)  The  new  test  can  be  successfully 
applied  to  a  blood  which  has  been  mixed  with  those  of  several  other  animals. 
(5)  We  have  in  this  test  the  most  delicate  means  hitherto  discovered  of  detecting 
and  testing  bloods,  and  consequently  we  may  hope  that  it  will  be  put  to  forensic 
use." 

Further  perfection  in  the  technic  of  the  precipitation  experiments 
can  be  found  in  a  paper  by  Nuttall  and  Inchley.* 

The  precipitinogen  is  capable  of  acting  as  an  antigen  and  the 
injection  into  animals  of  serum  containing  it  results  in  the  formation 
of  anti-precipitins. 

AGGLUTINATION 

Agglutination  is  a  phenomenon  of  infection  and  immunity  in 
which  the  serum  or  other  body  juice  of  the  infected  animal  so  acts 
upon  the  infecting  micro-organism  as  to  destroy  its  power  of  move- 
ment, and  cause  it  to  sediment  in  clusters  in  the  liquid  in  which  it 
is  suspended.  This  phenomenon  was  first  observed  by  Charrin 
and  Rogerf  in  the  course  of  experiments  with  Bacillus  pyocyaneus. 
They  found  that  when  bacillus  pyocyaneus  was  introduced  into  a 
test-tube  containing  the  diluted  serum  of  an  animal  infected  with  or 
immunized  against  it,  the  bacilli  ceased  their  active  movements, 
became  aggregated  in  clusters  and  settled  to  the  bottom  of  the  tube, 
leaving  the  supernatant  fluid  clear.  Observations  confirming  and 
enlarging  upon  the  subject  were  made  by  Metschnikoff,  J  Issaeff§ 
and  others.  Gruber  and  Durham  ||  made  an  elaborate  and  now 
classic  study  of  the  subject,  first  employing  the  term  "agglutina- 
tion" to  the  phenomenon,  and  "  agglutinins "  to  the  substances  in 
the  serum  by  which  it  might  be  brought  about.  They  found  that 
when  cholera  or  typhoid  bacilli  are  mixed  with  their  respective 
immune  serums,  the  organisms  lose  motility  and  become  aggre- 
gated in  clusters,  masses  or  "clumps."  They  further  showed  the 
reaction  to  be  specific  within  certain  limitations,  i.e.,  typhoid  im- 
mune serum  agglutinated  typhoid-like  bacilli  but  no  others,  etc., 
and  they  saw  in  the  phenomenon  a  practical  means  for  the  dif- 
ferentiation of  different,  closely  related  bacteria,  an  application  that 
has,  indeed,  become  a  useful  one* 

It  remained  for  Widal**  to  show  that  it  had  a  much  more  important 

*  "Journal  of  Hygiene,"  1904,  iv,  p.  201. 
f"Compte  rendu  de  la  Soc.  de  Biol.,"  1899,  P-  667. 
t  "Ann.  de  1'Inst.  Pasteur,"  1891,  v. 
§  Ibid.,  1893,  vn. 
"  •     ||  "Munchener  med.  Woch.,"  1896,  No.  9. 

**  "Societe  Medicale  des  Hopitaux,"  June  26,  1896, 


The  Agglutinins  123 

application,  in  that  the  micro-organism  being  known,  the  effect 
produced  by  a  serum  upon  it  would  be  an  indication  of  the  infec- 
tion of  the  animal  from  which  the  serum  was  secured.  The  first 
practical  application  was  made  in  connection  with  the  diagnosis  of 
typhoid  fever,  and  the  brilliant  success  attending  it  has  led  to  the 
test  being  known  as  the  "Widal  reaction." 

The  agglutinins  are  stable  substances  that  resist  drying  and  can 
be  kept  dry  and  active  for  years.  Widal  and  Sicard  found  that  they 
pass  with  difficulty  through  a  porcelain  filter  and  do  not  dialyze. 
They  are  precipitated  in  part  by  15  per  cent,  of  sodium  chloride  that 
throws  down  fibrinogen  and  further  precipitated  with  magnesium 
sulphate,  which  throws  down  globulins.  They  therefore  thought 
them  to  be  intimately  related  to  the  globulins  and  to  fibrinogen.  A 
temperature  of  6o°C.  diminishes  their  activity,  but  they  are  not 
destroyed  below  8o°C.  Sunlight  has  no  effect  upon  them. 

Metschnikoff  looks  upon  agglutination  as  preliminary  to  phagocy- 
tosis and  to  bacteriolysis,  and  thinks  it  the  effect  of  enzymes  in  the 
serum  preparing  and  clustering  the  bacteria  to  be  taken  up  by  the 
phagocytes.  Ehrlich*  finds  in  the  agglutinins  nothing  more  than 
receptors  of  what  he  denominates  the  II  order,  each  of  which 
possesses  a  zymophore  and  an  agglutinophore  group. 

Malvozf  found  that  the  addition  of  chemical  substances,  such  as 
safranin,  vesuvin,  and  corrosive  sublimate,  to  cultures  of  the  typhoid 
bacilli  would  cause  their  agglutination.  Typhoid  bacilli  retained 
on  the  Chamberland  filter  and  washed  for  a  long  time,  could  no 
longer  be  agglutinated,  and  were  found  to  have  lost  their  flagella 
and  to  be  without  motion.  This  led  Dineur,{  who  made  additional 
experiments,  to  conclude  that  agglutination  depended  upon  the 
flagella.  Malvoz§  found  that  bacteria  were  sometimes  agglutinated 
by  their  own  metabolic  products.  He  prepared  a  fresh  culture  of 
the  first  vaccine  of  the  anthrax  bacillus  by  thoroughly  distributing 
it  through  J£  cc.  of  distilled  water,  and  then  added  a  loopful  of  a 
six-day-old  culture.  After  standing  for  a  few  hours  typical  agglu- 
tinations were  observed  under  the  microscope. 

H.  C.  Ernst  and  Robey||  found  that  flagella  have  nothing  to  do 
with  agglutination,  which  subsequent  experiment  has  shown  to  be 
correct,  as  non-flagellated  bacteria  can  be  agglutinated  by  their 
respective  serums  quite  as  well  as  the  flagellated  forms. 

Bail,**  Joosft,  Eisenberg  and  Volltt  have  shown  that  all  of  the 
agglutinins  possess  haptophore  and  agglutinophore  groups,  either 
of  which  may  be  destroyed  without  the  other.  Thus  typhoid 

*  See  Nothnagel's  "Specielle  Pathologie  und  Therapie,"  1901,  vm. 

f  "Ann.  de  1'Inst.  Pasteur,"  1897,  No.  6. 

t  "Bull,  de  1'Acad.  de  Med.  de  Belgique,"  1898,  iv,  p.  705. 

§  "Ann.  de  1'Inst.  Pasteur,"  Aug.  25,  1899. 

||  "Trans.  Cong.  Amer.  Phys.  and  Surg.,"  1900,  p.  26. 
**  "Archiv  f.  Hyg.,"  1902,  XLII,  Heft  4. 
ft  "Zeitschr.  f.  Hyg.,"  1901,  xxxvi,  p.  422. 
it  Ibid.,  1902,  XL,  p.  155. 


1 24  Immunity 

agglutinative  serum  when  exposed  to  a  temperature  of  65°C. 
loses  the  agglutinophores,  and  no  longer  clumps  the  bacteria,  though 
it  retains  the  haptophores,  and  when  brought  into  contact  with  the 
bacteria  combines  with  them,  producing  no  agglutination,  but  pre- 
venting the  action  of  unheated  agglutinogenic  serum. 

Buxton  and  Vaughan*  found  that  bacteria  differ  both  in  their 
agglutinogenic  powers  and  their  agglutinability,  both  of  which  must 
be  taken  into  account  in  studying  the  subject. 

Theobald  Smithf  has  shown  that  there  are  two  kinds  of  agglutinins, 
one  of  which  acts  upon  the  bacteria  directly,  the  other  through 
the  flagella.  The  occurrence  of  these  two  bodies  explains  some  of 
the  incompatible  results  of  previous  experiments. 

The  reaction  is  one  of  the  most  delicate  known  to  us  for  the 
identification  of  bacteria.  It  is  so  specific  that,  in  the  case  of  many 
organisms,  it  is  even  possible  to  tell  from  what  original  source  they 
may  have  come,  and  always  to  tell  to  what  variety  they  belong. 
It  is,  moreover,  a  comparatively  simple  method  that  can  be  used  by 
physicians  with  little  technical  skill.  The  various  serums  necessary 
can  be  obtained  from  the  large  public  and  commercial  laboratories 
where  animals  immunized  against  various  cultures  can  always  be 
kept  on  hand  and  periodically  bled.  The  serums,  sealed  in  small 
tubes,  can  be  kept  an  almost  unlimited  length  of  time  and  shipped 
to  any  distance  ready  for  use  when  opened  and  diluted. 

There  is  no  uniform  technic  by  which  to  apply  the  test.  Scarcely 
any  two  laboratories  employ  the  same  method,  but  the  results  are 
uniform  and  the  method  to  be  employed,  provided  it  is  free  from 
error,  is  that  found  most  convenient  to  the  individual  operator. 

The  agglutination  test  now  subserves  two  important  functions: 
i,  the  diagnosis  of  any  infectious  disease,  provided  the  infecting  or- 
ganism be  at  hand;  2,  the  recognition  of  any  micro-organism,  provided 
specific  serum  be  at  hand. 

Technic  of  Agglutination  Tests 

If  possible,  a  culture  of  the  micro-organism,  grown  upon  agar-agar,  is  to  be 
selected  for  the  purpose.  A  good-sized  platinum  loopful  of  the  culture  is  taken 
up  and  distributed  as  uniformly  as  possible  throughout  a  few  cubic  centimeters 
of  distilled  water.  This  is  best  done  by  placing  the  water  in  a  test-tube  and  then 
rubbing  the  culture  upon  the  glass  just  above  the  level  of  the  fluid,  until  it  is 
thoroughly  emulsified,  permitting  it  to  enter  the  water  little  by  little  and,  finally, 
washing  it  all  down  into  the  fluid.  This  gives  a  distinctly  cloudy  fluid,  too  con- 
centrated to  use.  Of  this  one  adds  enough  to  each  of  a  series  of  watch-glasses 
or  test-tubes,  each  containing  an  equal  volume  of  distilled  water  (say  2  cc.), 
to  make  the  fluid  opalescent  by  reflected  light  though  transparent  by  trans- 
mitted light.  The  same  quantity  should  be  added  to  each,  so  that  they  form 
a  uniform  series.  The  patient's  blood  or  serum  is  next  diluted  and  added  so 
that  the  watch-glasses  or  tubes  receive  a  i :  10,  i :  20,  i :  30,  i :  40,  i :  50,  i :  60,  i :  80, 
1:100,  1:150,  1:200,  1:300,  or  a  laboratory  serum  of  high  agglutinative  value, 
1:1000,  1:2000,  1:5000,  i :  10,000,  1:50,000,  and  1:100,000. 

If  watch-glasses  are  used,  they  are  stood  upon  a  black  surface,  covered, 

*  "Jour.  Med.  Research,"  July,  1904. 
f  Ibid.,  1904,  vol.  x,  p.  89. 


The  Antitoxins  125 

and  examined  in  fifteen,  thirty,  and  sixty  minutes  by  simply  looking  at  the  dark 
surface  through  the  fluid.  If  agglutination  occur,  the  original  opalescence  gives 
place  to  a  slightly  curdy  appearance,  as  the  uniformly  suspended  bacteria 
aggregate  in  clumps. 

If  test-tubes  are  employed,  they  are  best  observed  by  tilting  them  and  look- 
ing through  a  thin  layer  of  the  contained  fluid  at  a  dark  surface  or  at  the  sky. 
In  either  case  the  flocculent  collections  of  agglutinated  bacteria  can  be  seen. 

The  test  can  also  be  made  and  observed  under  the  microscope  by  the  hanging- 
drop  method,  but  in  working  with  such  small  quantities  much  of  the  accuracy 
of  the  technic  is  apt  to  be  lost. 

Some  knowledge  is  required  in  order  to  form  correct  deductions  from  the  ex- 
periments. Thus,  with  typhoid  bloods,  the  agglutination  of  the  typhoid  bacillus 
usually  occurs  within  an  hour  in  dilutions  of  i :  50,  but  the  agglutinability  of 
the  culture  employed  should  be  known  before  the  experiment  is  undertaken. 

Similarly,  when  the  method  is  employed  for  the  differentiation  of  bacteria  the 
agglutinative  value  of  the  serum  should  be  known  to  begin  with. 

The  agglutinins  are  capable  of  acting  as  antigens  and  when  in- 
jected into  animals  effect  reactions  followed  by  the  formation  of 
antibodies  inhibiting  their  own  activity. 

ANTITOXINS 

Antitoxins  are  immunity  products  by  which  the  injurious  actions 
of  toxins  are  annulled.  In  the  synopsis  of  immunity  experiments 
already  given,  the  history  of  the  discovery  and  development  of  the 
antibodies  has  been  outlined,  together  with  references  to  the 
original  contributions  in  which  they  were  made  public. 

In  the  section  upon  the  "Explanation  of  Immunity"  we  have 
seen  that  the  best  mode  of  accounting  for  the  occurrence  of  antitoxins 
is  afforded  by  Ehrlich  in  the  lateral-chain  theory.  He  regards  them 
as  cell  haptophiles — receptors — that  are  formed  in  excess  of  the  re- 
quirements, by  cells  frequently  stimulated  by  the  presence  of  bacterial 
products  possessing  adapted  haptophores.  The  receptors  are  under 
normal  conditions  engaged  in  maintaining  the  proper  nutrition  of 
the  cell;  under  abnormal  conditions  (as  when  preempted  by  the  inert 
or  injurious  haptophores  of  the  bacterial  products)  are  obliged  to 
increase  in  number  to  compensate  for  the  damage  done  the  cell. 
Antibody  formation  can  be  induced  only  by  antigens  or  bodies 
that  bear  a  resemblance  to  the  normal  nutrient  substances  absorbed 
by  the  cells  in  that  they  are  provided  with  haptophore  groups 
corresponding  with  the  haptophile  groups  of  the  cells  and  so  adapted 
for  union  with  them.  Mineral  and  alkaloidal  substances  have 
no  such  adaptations,  but  bacterial  products,  the  toxalbumins 
of  various  higher  plants,  venoms,  enzymes,  and  other  protein  com- 
binations have.  The  possession  of  the  haptophile  groups  determines 
whether  or  not  the  cell  can  stimulate  antibody  formation,  and  the 
ability  to  produce  antibodies  shows  the  existence  of  the  haptophore 
groups. 

The  attachment  of  the  haptophore  groups  to  the  cells  is  usually 
shown  by  morbid  action  of  the  cells  in  cases  where  there  are  as- 
sociated toxophore  and  toxophile  groups,  as  in  the  case  of  thebacterio- 


126  Immunity 

toxins,  but  may  not  be  discovered  if  there  are  none.  The  combina- 
tion of  the  toxin-haptophores  with  the  cell-haptophiles  can  be 
demonstrated  in  the  test-tube  by  crushing  the  cerebral  substance  of 
a  rabbit,  and  adding  tetanus  toxin.  The  toxin  becomes  fixed  by 
combination  with  the  cell  haptophiles  or  receptors,  loses  its  further 
combining  powers  and  fails  to  affect  animals  into  which  it  is  sub- 
sequently injected.  The  increased  formation  of  receptors  in  con- 
sequence of  repeated  stimulation  has  been  shown  by  the  effect  of 
abrin  upon  the  conjunctiva.  If  dropped  into  one  eye  until  the 
conjunctiva  is  thoroughly  immune  against  its  action,  the  cells  of 
this  eye  develop  a  greatly  increased  capacity  for  absorbing — i.e., 
fixing — the  abrin  as  compared  with  those  of  the  other  eye.  Thus  if 
the  two  conjunctival  membranes  be  dissected  out  and  a  certain 
quantity  of  abrin  triturated  with  each,  the  haptophiles  of  the  cells 
of  the  immunized  membrane  fix  the  poison  so  that  it  is  no  longer  able 
deleteriously  to  affect  animals,  while  no  such  effect  takes  place  with 
the  other  membrane. 

The  ability  to  stimulate  the  formation  of  antibodies  is  entirely 
independent  of  any  toxic  action  and  is  entirely  the  work  of  the  hap- 
tophiles. This  is  best  shown  in  the  fact  that  diphtheria  toxin  that 
has  been  heated  or  otherwise  manipulated  until  its  toxic  action  is 
lost,  still  retains  the  power  of  combining  with  antitoxin,  or  of 
producing  antibodies. 

The  cells  furnishing  the  haptophile  groups  or  receptors  whose 
presence  in  the  blood  gives  it  its  antitoxic  quality  vary  in  number  or 
quality  in  different  animals.  Thus,  in  the  warm-blooded  animals 
the  rapidity  with  which  tetanus  toxin  is  anchored  to  the  cells  of 
the  central  nervous  system  seems  to  indicate  that  those  cells,  if 
not  the  only  cells  in  the  body  passing  the  adapted  receptors  by 
which  it  is  anchored,  are  the  chief  cells  by  which  it  is  absorbed. 
In  the  alligator,  however,  other  cells  seem  to  fix  the  toxin  before  it 
reaches  or  connects  with  those  of  the  nervous  system,  so  that  the 
alligator,  though  immune  against  the  action  of  the  toxin,  is  able  to 
make  antitoxin  as  well  as  susceptible  animals. 

Each  introduction  of  appropriate  antibody  forming  substance 
is  followed  by  an  outpouring  of  the  antibody  far  in  excess  of  what 
would  neutralize  it,  so  that  after  a  systematic  treatment  has  been 
carried  out  for  some  time,  the  neutralizing  value  of  the  blood  may  be 
a  thousand  times  what  would  be  necessary  to  neutralize  the  total 
quantity  of  active  substance  introduced  into  the  animal. 

Each  antibody  is  specific  in  action,  as  must  be  evident  from  its 
mode  of  formation.  Should  it  be  found,  however,  that  several  active 
bodies  possessed  haptophore  groups  of  identical  structure,  the  anti- 
body formed  by  any  of  them  might  be  found  to  possess  common 
neutralizing  powers  for  all. 

The  animal  whose  blood  contains  antibodies  enjoys  immunity 
from  the  active  body  by  which  they  were  formed  only  so  long  as 


The  Antitoxins  127 

they  are  present.  In  some  cases,  however,  animals  that  have 
been  long  subjected  to  the  immunization  treatment,  and  whose  blood 
contains  large  quantities  of  free  antitoxin,  unexpectedly  become 
abnormally  sensitive  (hypersensitivity)  to  the  toxin,  and  may  die 
after  receiving  a  very  small  dose.  This  may  be  attributed  to  a 
difference  in  the  combining  activity  of  the  receptors  attached  to  the 
cells,  and  those  separated  and  free  in  the  serum.  If  the  former 
developed  a  greater  affinity  for  the  toxin  than  the  latter,  it  would 
unite  with  them  by  preference  and  intoxication  ensue.  If  the  treat- 
ment by  which  the  antitoxins  are  produced  is  interrupted,  they  im- 
mediately begin  to  lessen  in  quantity,  and  eventually  disappear. 
Their  occurrence  in  the  blood  determines  that  they  shall  be  found 
in  all  the  body  juices,  though  in  varying  quantity. 

Their  chemical  composition,  which  experiment  shows  to  be  of 
protein  nature,  determines  that  when  practical  use  is  to  be  made 
of  them,  they  must  not  be  administered  by  the  stomach,  as  diges- 
tion is  usually  followed  by  their  destruction.  In  infants,  the  protein 
digestion  being  feeble,  antitoxins  pass  from  the  mother's  milk  to 
the  blood  of  the  sucking  offspring  without  digestion,  but  the  ad- 
ministration of  antitoxins  by  this  method  at  later  periods  of  life  is 
followed  by  effects  too  uncertain  to  be  depended  upon.  For  practical 
therapeutic  purposes,  therefore,  the  administration  must  always  be 
made  hypodermically  or  intravenously. 

Diphtheria  Antitoxin. — This  was  first  utilized  for  practical 
therapeutic  purposes  by  Behring.*  As  usually  prepared  by  the 
administration  of  the  toxin,  it  is  essentially  an  antitoxin  and  has 
no  destructive  action  upon  the  diphtheria  bacilli.  In  therapeutics 
it  is  employed  to  neutralize  or  "fix"  the  toxin  circulating  in  the 
blood,  not  to  destroy  the  bacilli,  or  to  effect  the  regeneration  of  the 
tissues  injuriously  acted  upon  by  the  toxin.  Martin  is  of  the 
opinion  that  such  purely  antitoxic  serums  are  inferior  to  those  con- 
taining other  immunity  products,  such  as  bacteriolysins,  and  recom- 
mends that  the  whole  culture  instead  of  the  filtered  culture  be  used 
in  the  immunization  of  the  animal.  If  this  is  done,  the  bacteriolytic 
effect  is  added  to  the  antitoxic  effects  of  the  serum. 

The  serum  may  be  used  to  prevent  or  to  cure  diphtheria. 

The  antitoxin  is  commercially  manufactured  at  present  by  im- 
munizing horses  against  increasing  quantities  of  diphtheria  toxin 
until  the  proper  degree  of  immunity  has  been  attained,  then  with- 
drawing the  antitoxic  blood.  The  details  are  as  follows: 

I.  The  Preparation  of  the  Toxin. — The  toxic  metabolic  products  of  tha 
Bacillus  diphtherias  are  for  the  most  part  freely  soluble,  and  are  therefore  best 
prepared  in  cultures  grown  in  fluid  media.  The  medium  best  adapted  to  the 
purpose  is  that  lecommended  by  Theobald  Smith,  f 

To  make  it,  the  usual  meat  infusion  receives  the  addition  of  a  culture  of 

*  "Deutsche  med.  Wochenschrift,"  1890,  Nos.  49  and  50;  "Zeitschrift  fiir 
Hygiene,"  etc.,  1892,  xn,  p.  i;  "Die  Blutserumtherapie,"  Berlin,  1902'. 
f  "Journal  of  Experimental  Medicine,"  May  and  July,  1899,  p.  373. 


1 28  Immunity 

Bacillus  coli,  and  is  stood  in  a  warm  place  overnight.  The  colon  bacilli  ferment 
and  remove  the  muscle  and  other  sugars.  The  infusion  is  then  made  into 
bouillon,  titrated  so  that  the  reaction  equals  +  T.I  when  tested  with  phenolph- 
thalein.  It  then  receives  an  addition  of  0.2  per  cent,  of  dextrose,  and  is  sterilized 
in  the  autoclave.  To  secure  the  best  toxic  product,  the  bacilli  at  hand  must  be 
carefully  studied  and  that  naturally  possessing  the  strongest  toxicogenic  power 
employed  for  the  cultures.  The  greatest  toxicity  seems  to  develop  between  the 
fifth  and  seventh  days.  If  the  culture  is  permitted  to  remain  in  the  incubating 
oven  beyond  this  period,  the  toxin  gradually  is  transformed  to  toxoid  and  its 
activity  declines.  The  fatal  dose  for  a  250-300  gram  guinea-pig  should  be  about 
o.ooi  cc.  given  hypodermically. 

II.  The  Immunization  of  the  Animals. — All  commercial  manufacturers  of 
diphtheria  antitoxic  serums  now  use  horses,  as  recommended  by  Roux,  instead 
of  the  sheep,  dogs,  and  goats  with  which  the  earlier  investigators  worked.  The 
horse  is  readily  immunized,  gives  an  abundant  supply  of  blood  which  clots  readily 
and  yields  a  beautiful  clear  amber  serum. 

The  horse  selected  should  be  in  perfect  health,  and  should  be  tested  with 
mallein  and  tuberculin  to  avoid  obscure  glanders  and  tuberculosis. 

A  small  dose  of  the  toxic  bouillon — say  o.i  cc. — should  be  given  in  the  begin- 
ning, as  one  occasionally  finds  exceptionally  susceptible  animals  that  will  suc- 
cumb to  larger  doses.  If  a  marked  local  and  general  reaction  follows,  it  may  be 
better  to  try  another  animal.  If  no  reaction  is  brought  about,  the  immunization 
is  carried  on  as  rapidly  as  possible.  The  toxin  is  injected  hypodermatically 
into  the  tissues  of  the  neck,  the  skin  being  thoroughly  cleaned  and  disinfected 
before  each  injection.  The  doses  are  cautiously  increased  and  may  often  be 
doubled  each  day.  If  any  unfavorable  symptoms  arise,  treatment  must  be  in- 
terrupted for  a  day  or  two.  The  animal  yields  good  antitoxic  serum  when  it 
can  endure  several  doses  of  500  cc.  of  the  strong  toxin  mentioned  above. 

IIL  Bleeding. — When  the  withdrawal  of  a  small  quantity  of  blood  by  a 
hypodermic  needle  introduced  into  the  jugular  vein  shows  that  the  serum  con- 
tains a  maximum  antitoxic  strength  (300  to  1000  units  per  cubic  centimeter), 
the  horse  is  ready  to  bleed.  Some  horses  can  be  bled  without  resistance,  but 
most  of  them  require  to  be  fastened  in  appropriate  stocks.  The  blood  is  taken 
from  the  jugular  vein,  which  is  superficial,  of  large  size,  and  easily  accessible. 
The  skin  is  carefully  shaved  over  an  area  about  9  square  inches  in  extent,  thor- 
oughly disinfected.  A  small  incision  is  made  over  the  .center  of  the  vein,  which 
is  made  prominent  by  pressure  at  the  base  of  the  neck,  and  the  point  of  a  small 
sterile  trocar  being  inserted  in  the  incision  through  the  skin,  it  is  directed  obliquely 
upward  into  the  vein.  The  blood  is  allowed  to  flow  through  a  sterile  tube 
attached  to  the  cannula  into  sterile  bottles  prepared  to  receive  it.  A  large  horse 
may  furnish  7  to  9  liters;  small  horses,  5  to  7  liters. 

IV.  Preparation  of  the  Serum. — The  blood  is  stood  away  in  a  cool  place  until 
the  clot  retracts  after  coagulation  and  the  clear  serum  separates.     The  serum 
is  then  withdrawn  under  strict  aseptic  precautions.     It  is  variously  prepared 
for  the  market.     Some  manufacturers  bottle  it  without  any  added  preservative; 
some  add  a  crystal  of  thymol;  some  Pasteurize  it;  some  add  carbolic  acid;  some 
add  trikresol. 

The  plain  serum  would  be  ideal,  but  the  danger  of  subsequent  contamination 
through  careless  treatment  makes  it  rather  better  to  have  an  antiseptic  added. 
Trikresol  is  probably  the  most  satisfactory  of  these,  though  it  throws  down  a 
precipitate  that  necessitates  the  filtration  of  the  product,  and  leaves  the  serum 
slightly  opalescent. 

V.  Determining  the  Potency  of  the  Serum. — The  potency  of  the  serum  is 
expressed  as  so  many  "immunizing  units."     Only  one  method  of  testing  is 
in  use  at  the  present  time,  though  to  understand  it,  it  seems  wise  to  mention 
the  original  method  from  which  it  was  derived. 

(A)  Behring's  Method. — Behring's  unit  was  an  arbitrary  standard  chosen  in 
consequence  of  certain  conditions  existing  at  the  time  it  was  devised.  It  is 
difficult  to  understand  apart  from  the  circumstances  governing  its  creation,  but 
may  be  defined  as  "  Ten  times  the  least  quantity  of  antitoxin  serum  that  will  protect 
a  standard  (300  gram)  guinea-pig  against  ten  times  the  least  certainly  fatal  dose  of 
toxic  bouillon" 

The  method  of  determining  it  is  not  difficult  to  those  skilled  in  laboratory 
technic,  and  is  as  follows: 

i.  Determine  accurately  the  least  certainly  fatal  dose  of  a  sterile  diphtheria 
toxic  bouillon  for  a  standard  guinea-pig. 


The  Antitoxins  129 

2.  Determine  accurately  the  least  quantity  of  the  serum  that  will  protect 
the  guinea-pig  against  ten  times  the  above  determined  least  fatal  dose  of  toxin. 

3.  Express  the  required  dose  of  antitoxic  serum  as  a  fraction  of  a  cubic  centi- 
meter and  multiply  by  10;  the  result  is  one  unit. 

Example:  It  is  found  that  o.oi  cc.  of  a  toxic  bouillon  kills  at  least  9  out  of  10 
guinea-pigs,  and  is  therefore  the  least  certainly  fatal  dose.  Guinea-pigs  receive 
ten  times  this  dose  of  the  toxic  bouillon  plus  varying  quantities  of  the  serum  to 
be  tested,  measured  by  dilution—  say  Hooo  cc.,  Hsoo  cc.,  3^000  cc.  The 

lied  b 


first  two  live.     The  fraction  >1}500  is  now  multiplied  by  10;  ^3500  X  10 

=  i  unit.     So  we  find  that  each  cubic  centimeter  of  the  serum  contains  250 

units. 

This  method  would  be  satisfactory  were  it  not  for  certain  variations  in  the 
toxic  bouillon  by  which  the  strength  is  worked  out.  Ehrlich,*  in  an  elaborate 
investigation  of  these  changes,  has  clearly  proved  that  an  ever-changing  toxin 
cannot  be  a  satisfactory  standard,  because  it  does  not  possess  uniform  combining 
affinity  for  the  antitoxin.  He  shows  by  a  labored  scheme  that  the  toxicity  of 
the  bouillon  is  no  index  to  its  antitoxin-combining  power,  which,  of  course,  must 
be  the  foundation  of  the  test.  The  toxin,  under  natural  conditions,  is  changed 
with  varying  rapidity  into  toxoids,  of  which  he  demonstrates  three  groups  — 
prototoxoids,  syntoxoids,  and  epitoxoids.  The  epitoxoids  have  a  greater  anti- 
toxin-combining power  than  the  toxin  itself,  yet  have  no  toxic  action  upon  the 
guinea-pigs,  hence  cause  confusion  in  the  results. 

To  secure  a  satisfactory  measure  of  the  antitoxic  strength  of  a  serum,  it  is 
therefore  more  important  to  first  determine  the  antitoxin-combining  power  of 
the  toxin  or  toxic  bouillon  to  be  used  than  to  determine  its  guinea-pig  fatality, 
and  this  is  what  Ehrlich  endeavors  to  do. 

(B)  Ehrlich'  s  Method.  —  In  this  method  the  unit  is  the  same  as  in  Behring's 
method,  but  its  determination  is  arrived  at  by  a  very  important  modification  of 
the  method,  by  which  the  standard  of  measurement  is  a  special  antitoxin  of 
known  strength,  by  which  the  antitoxin-combining  power  of  the  test  toxic  bouil- 
lon is  first  determined.  Ehrlich  began  by  determining  the  antitoxic  value  of  a 
serum  as  accurately  as  possible  by  the  old  method,  and  then  used  that  serum  as 
the  standard  for  all  further  determinations.  The  serum  was  dried  in  a  vacuum, 
and  two  grams  of  the  dry  powder  were  placed  in  each  of  a  large  number  of 
small  vacuum  tubes,  connecting  with  a  small  bulb  of  phosphoric  anhydride. 
In  this  way  the  standard  powder  was  protected  from  oxygen,  water,  and  other 
injurious  agents  by  which  variations  in  its  strength  could  be  initiated.  Periodic- 
ally one  of  these  tubes  was  opened  and  the  contained  powder  dissolved  in  200 
cc.  of  a  mixture  of  10  per  cent,  aqueous  solution  of  sodium  chloride  and  glycerin. 
The  subsequent  calculations  are  all  based  upon  the  strength  of  the  antitoxin 
powder.  In  Ehrlich's  first  test  serum  i  gram  of  the  dry  powder  represented 
1700  units.  Of  the  solution  mentioned,  i  cc.  represented  17  units;  ^7  cc., 
one  unit. 

Having  by  dilution  —  i  cc.  of  the  first  dilution  in  17  of  water  —  secured  the 
standard  unit  of  antitoxin  in  a  convenient  bulk  for  the  subsequent  manipulations, 
it  is  mixed  with  varying  quantities  of  the  toxic  bouillon  to  be  used  for  testing  the 
new  serums,  until  the  least  quantity  is  determined  that  will  cause  the  death  of  a 
250  gram  guinea-pig  in  exactly  four  days,  when  carefully  injected  beneath  the 
skin  of  the  animal's  abdomen.  This  quantity  of  toxin  is  the  test  dose.  If  the 
toxic  bouillon  was  "normal"  in  constitution,  it  should  represent  100  of  the  least 
certainly  fatal  doses  that  formed  the  basis  of  the  old  method  of  testing,  but  as 
toxic  bouillons  contain  varying  quantities  of  toxoids  it  may  equal  anywhere 
from  fifty  to  one  hundred  and  fifty  times  that  dose. 

The  test  dose  of  toxic  bouillon,  having  been  determined,  remains  invariable 
throughout  the  test  as  before,  the  serum  to  be  tested  for  comparison  with  the 
standard  being  modified.  The  calculation  is,  however,  different  because  the 
guinea-pig  is  receiving,  not  ten  times,  but  more  nearly  one  hundred  times  the 
least  fatal  dose,  and  the  quantity  of  the  antitoxic  serum  that  preserves  life 
beyond  the  fourth  day  is  itself  the  unit. 

Example:  The  sample  of  serum  issued  as  the  standard  contains  17  units  per 
cubic  centimeter.  Serum  i  cc.  +  water  16  cc.  =  i  cc.  is  the  unit,  i  cc.  of 
the  dilution  containing  one  antitoxic  unit  is  mixed  with  o.oi,  0.025,  0.05,  0.075, 
o.i  cc.  of  the  toxic  bouillon.  All  the  animals  receiving  less  than  o.i  cc.  live. 

*  "Klinisches  Jahrbuch,"  1897. 


130  Immunity 

A  new  series  is  started,  and  the  guinea-pigs  all  weighing  exactly  250  grams, 
receive  i  unit  of  the  antitoxin  plus  toxic  bouillon  0.08,  0.09,  0.095,  °-°97,  o-1? 
o.i i,  0.12,  etc.  It  is  found  that  all  receiving  more  than  0.097  die  in  four  days, 
but  that  the  animal  receiving  that  dose,  though  very  ill,  lives  longer.  The 
test  dose  may  then  be  assumed  to  be  o.i,  or  it  may  be  calculated  more  closely 
if  desired. 

To  test  the  serum  itself,  guinea-pigs  weighing  exactly  250  grams  are  now 
all  given  toxic  bouillon  o.i  cc.plus  varying  quantities  of  the  serum — 3^oo>  Moo* 
Moo>  etc-  All  live  except  those  receiving  less  than  ^oo>  which  die  about  or  on 
the  fourth  day.  The"  serum  can  then  be  assumed  to  have  400  units  per  cubic 
centimeter  unless  it  be  desired  to  test  more  closely. 

Standard  test  serums  for  making  tests  of  antitoxic  serums  by  the 
Ehrlich  method  were  first  shipped  at  small  expense  from  the  Kaiser- 
liches  Institut  fur  Serum-Therapie  at  Hochst-on-the-Main.  At 
present  the  Hygienic  Laboratory  of  the  United  States  Public  Health 
Service  has  legal  control  of  the  manufacture  of  therapeutic 
serums  and  kindred  products  in  the  United  States,  issuing  licenses 
to  those  engaged  in  legitimate  manufacture,  and  furnishing  a 
standard  test  serum,  similar  to  that  of  Ehrlich,  to  those  entitled  to 
receive  it. 

A  full  description  of  "The  Immunity  Unit  for  Standardizing 
Diphtheria  Antitoxin,"  by  M.  J.  Rosenau,  Director  of  the  Hygienic 
Laboratory,  can  be  found  in  Bulletin  No.  21  of  the  U.  S.  Public 
Health  and  Marine  Hospital  Service,  Washington,  1905. 

As  the  quantity  to  be  injected  at  each  dose  diminishes  according 
to  the  number  of  units  per  cubic  centimeter  the  serum  contains,  it 
is  of  the  highest  importance  that  therapeutic  serums  be  as  strong 
as  possible.  Various  t  methods  of  concentration  have  been  sug- 
gested. Bujwid*  and  H.  C.  Ernstf  found  that  when  an  antitoxic 
serum  is  frozen  and  then  thawed,  it  separates  into  two  layers,  the 
upper  stratum  watery,  the  lower  yellowish,  the  antitoxic  value  of 
the  yellowish  layer  being  about  three  times  that  of  the  original 
serum,  the  upper  layer  consisting  chiefly  of  water. 

The  most  satisfactory  method  of  securing  a  useful  concentration 
is  by  the  employment  of  the  globulin  precipitation  as  recommended 
by  Gibson, %  which  is  briefly  as  follows:  The  diluted  citrated  plasma 
is  precipitated  with  an  equal  volume  of  saturated  ammonium  sul- 
phate solution  and  the  antitoxic  proteins  separated  by  extracting 
the  precipitate  with  saturated  sodium  chloride  solution.  The  soluble 
antitoxic  proteins  are  then  reprecipitated  from  the  saturated  sodium 
chloride  solution  with  acetic  acid.  This  filtered  precipitate  is  then 
partially  dried  between  filter-papers  and  dialyzed  in  running  water. 
This  yields  a  final  product  which  when  dried  in  vacuo  is  readily  solu- 
ble in  salt  solution  and  is  free  from  many  of  the  offensive  substances 
in  the  horse  serum.  Steinhardt  and  Bauzhaf  §  found  that  the  thera- 

*"Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Sept.,  1897,  Bd.  xxn,  Nos.  10  and  n, 
p.  287. 

•"  Jour.  Boston  Soc.  of  Med.  Sci.,"  May,  1898,  vol.  n,  No.  8,  p.  137. 

j  "Jour.  Biol.  Chem.,"  i,  p.  161;  in,  p.  253. 

§  "Jour.  Infectious  Diseases,"  March,  1908,  vol.  n,  pp.  202  and  264. 


The  Antitoxins  131 

peutic  value  of  the  plasma  was  not  appreciably  impaired  through  the 
process  of  eliminating  the  albumins  and  other  non-antitoxic  proteins 
by  the  salting  out  methods  employed,  and  the  final  dialyzation  of 
the  concentrated  product,  thus  disproving  the  objection  of  Cruveil- 
hier*  on  this  point. 

Tetanus  antitoxin  was  first  prepared  by  Behring  and  Kitasato.f 
It  can  be  employed  for  the  prevention  or  cure  of  tetanus.  For  the 
former  purpose,  hypodermic  injections  of  the  serum  may  be  given  in 
cases  with  suspicious  wounds,  or  the  wounds  may  be  dusted  with  a 
powder  made  by  pulverizing  the  dried  serum.  For  treatment  the 
serum  must  be  administered  in  frequently  repeated  large  doses  by 
hypodermic  or  intravenous  injection.  The  results  are  less  brilliant 
than  those  attained  with  diphtheria  antitoxin  because  of  the  avidity 
with  which  the  cells  of  the  central  nervous  system  take  up  the  tetanus 
toxin,  and  the  firmness  of  the  union  formed.  An  analysis  of  a  great 
number  of  cases  has,  however,  shown  that  the  recoveries  following 
the  free  administration  of  the  serum  exceed  those  effected  by  other 
methods  of  treatment  by  about  40  per  cent. 

By  the  gradual  introduction  of  tetanus  toxin  Behring  and  Kita- 
satoj  have  been  able  to  produce  a  powerful  antitoxic  substance  in 
the  blood  of  animals. 

The  method  of  obtaining  tetanus  antitoxic  serum  is  like  that 
employed  for  securing  diphtheria  antitoxic  serum  (q.v.). 

Madsen§  found  that  for  each  of  the  specific  poisons,  tetanolysin 
and  tetanospasmin,  a  specific  antitoxin  is  produced,  the  one  annul- 
ling the  convulsive,  the  other  the  hemolytic,  properties  of  the  toxin. 
The  usual  therapeutic  serums  contain  both  of  these. 

Different  standards  for  measuring  the  strength  of  the  tetanus 
toxin  and  different  definitions  of  the  unit  of  measurement  are 
given  in  different  countries,  so  that  great  confusion  and  dissatis- 
faction were  experienced  until  a  special  committee  of  the  Society 
of  American  Bacteriologists  met  in  New  York,  Dec.  27  and  28, 
1906,  and  in  collaboration  with  the  United  States  Public  Health  and 
Marine  Hospital  Service,  Hygienic  Laboratory,  formulated  a 
standard  unit  which  has  become  the  legal  unit  of  measurement  for 
the  United  States.  It  is  thus  defined: 

"The  immunity  unit  for  measuring  the  strength  of  tetanus 
antitoxin  shall  be  ten  times  the  least  quantity  of  antitetanic  serum 
necessary  to  save  the  life  of  a  350-gram  guinea-pig  for  ninety-six 
hours  against  the  official  test  dose  of  a  standard  toxin  furnished  by 
the  Hygienic  Laboratory  of  the  Public  Health  and  Marine  Hospital 
Service."  The  unit  is  thus  officially  defined,  Oct.  25,  1907,  in 
Treasury  Circular  No.  61. 

Testing  tetanus  antitoxic  serums  immediately  became  a  matter 

*  "Ann.  de  1'Inst.  Pasteur,"  1904,  xvm,  p.  249. 
t  "Deutsche  med.  Wochenschrift,"  1890,  No.  49. 
t  Ibid. 
§  "Zeitschrift  fur  Hygiene,"  1899,  xxxin,  p.  239. 


132  Immunity 

of  great  simplicity.  The  governmental  laboratory  furnishes  the 
"test  toxin"  whose  strength  is  guaranteed,  and  what  follows  is  a 
simple  matter  of  dilution,  admixture  with  the  serum  to  be  tested, 
and  the  injection  of  animals  that  are  carefully  observed  for  a  few 
days. 

The  entire  subject,  historical,  theoretical,  and  practical,  is  treated 
in  Bulletin  No.  43,  1908,  of  the  Hygienic  Laboratory  upon  "The 
Standardization  of  Tetanus  Antitoxin,"  by  Rosenau  and  Anderson. 

Antivenene  or  Anti-venomous  Serum. — This  was  discovered 
by  Phisalix  and  Bertrand*  and  made  practical  for  therapeutic 
purposes  by  Calmette.  f  Calmette  found  that  cobra  venom  con- 
tained two  principles,  one  of  which,  labile  in  nature  and  readily 
destroyed  by  heat,  was  destructive  in  action  upon  the  tissues  with 
which  it  came  into  direct  contact;  the  other,  stable  in  nature,  was 
death-dealing  through  its  action  upon  the  respiratory  centers.  By 
heating  the  venoms  and  thus  destroying  the  irritative  principle, 
he  was  able  to  immunize  animals  against  the  other,  which  he  looked 
upon  as  the  important  element  of  the  venom.  The  immunized 
animals  furnished  an  anti-serum,  which  entirely  annulled  the  effect 
of  the  toxin  (modified  venom)  used  in  treating  them.  This  serum 
was  found  to  protect  rabbits  and  other  animals  against  both  modi- 
fied and  unmodified  cobra  venom,  and  was  used  successfully  in 
the  treatment  of  a  number  of  human  beings  who  had  been  bitten 
by  cobras.  Calmette,  however,  erroneously  concluded  that  be- 
cause in  most  venoms  studied  he  was  able  to  find  a  larger  or  smaller 
proportion  of  the  respiratory  poison,  it  constituted  the  essential 
element  of  the  venom  to  be  antagonized.  Arguing  from  this  stand- 
point, he  recommended  his  antivenene  in  all  cases  of  snake-bite, 
regardless  of  the  variety  of  serpent.  C.  J.  Martini  and  others 
showed  that  Calmette  was  wrong,  and  that  his  antivenene  was 
useless  in  the  treatment  of  the  bites  of  the  Australian  serpents, 
and  the  experiments  of  the  author  have  shown  it  to  be  useless  in  the 
treatment  of  the  bites  of  the  American  snakes.  In  the  venoms  of  our 
snakes — the  rattlesnake,  copper-head,  and  moccasin — the  poison 
is  essentially  locally  destructive  in  action,  the  fatal  influence  upon 
the  respiratory  centers  being  of  secondary  importance.  Flexner  and 
Noguchi,§  Noguchi||  and  Madsen  and  Noguchi,**  however,  ap- 
plied Ehrlich's  principle  to  the  investigation,  destroyed  the  toxo- 
phorous  group  of  the  venom  molecules,  and  succeeded  in  producing 
an  anti-serum  useful  in  antagonizing  the  active  principle — hemor- 
rhagin — -of  the  Crotalus  venom. 

*"Compt.  rendu  de  1'Acad.  des  Sciences  de  Paris,"  Feb.  5,  1894,  cxvm, 

P-  356- 

f  "Compt.  rendu  de  la  Soc.  de  Biol.  de  Paris,"  Feb.  10,  1894,  10  Series,  i, 
p.  i 20. 

J  "Intercolonial  Medical  Journal  of  Australia,"  1897,  n,  p.  537. 

§  "Journal  of  Experimental  Medicine,"  1901-1905,  vi,  p.  277. 

||  Ibid.,  1906,  vin,  p.  614. 
**Ibid.,  1907,  ix,  p.  18. 


The  Cytotoxins  133 

Antivenene  is  useful  in  the  treatment  of  cobra  invenomation, 
as  Calmette  has  shown  by  cases  treated  in  his  own  laboratory. 
The  serums  of  Noguchi  and  others  are  equally  useful  in  their  re- 
spective invenomations,  but  the  opportunity  for  successfully  em- 
ploying antivenenes  is  very  small.  Few  persons  are  bitten  where  the 
remedy  is  at  hand,  and  the  effects  of  venom  of  all  kinds  are  so  rapid 
that  immediate  treatment  is  required.  In  India  and  a  few  other 
reptile  infected  countries,  as  well  as  in  zoological  gardens  where  ven- 
omous serpents  are  kept,  and  in  laboratories  where  the  snakes  are 
kept  for  experimental  purposes,  it  is  well  to  be  provided  with  a 
supply  of  the  serum,  but  it  has  no  wide  sphere  of  usefulness. 

CYTOTOXINS 

Cytotoxins  are  immunity  products  that  exert  a  specific  destructive 
action  upon  cellular  antigens.  They  are  essentially  cell-dissolving 
products  of  immunity.  The  solution  of  the  cells,  of  whatever  kind, 
takes  place  through  the  complement,  native  to  the  blood,  fixed  to 
the  cells  by  the  specific  amboceptor.  The  complement  is  pre- 
sumably always  the  same  and  is  "present  in  all  normal  blood;  the 
amboceptor  is  an  " immune  body"  susceptible  of  artificial  produc- 
tion or  increase,  and  specifically  differs  according  to  the  particular 
cell  through  whose  antigenic  activity  it  was  produced. 

Hemolysis. — The  phenomena  of  hemolysis  or  the  solution  of 
erythrocytes,  caused  by  heterologous  serums  were  first  studied  by 
Creite*  and  Landois,|  who  studied  hemoglobinuria  following 
transfusion.  Subsequent  observations  were  made  upon  corpus- 
cular agglutination  and  solution  by  venoms  by  Mitchell  and 
Stewart |  and  by  Flexner  and  Noguchi §,  and  upon  the  effects 
upon  corpuscles  of  warm-blooded  animals,  of  the  poisonous 
serum  of  certain  eels  by  Mosso,||  Camus  and  Gley,**  and  Kossel.tt 
The  serious  consideration  of  the  subject  was,  however,  de- 
ferred until  Belfanti  and  Carbone|J  showed  that  if  horses  were 
injected  with  red  corpuscles  of  rabbits,  the  serum  thereafter 
obtained  from  the  horses  would  be  toxic  for  rabbits;  Bordet§§ 
had  shown  that  the  serum  of  guinea-pigs  injected  several  times  with 
3  to  5  cc.  of  the  defibrinated  blood  of  rabbits  acquired  the  property 
of  rapidly  dissolving  the  red  corpuscles  of  the  rabbit  in  a  test-tube, 
and  Ehrlich  and  Morgenroth||||  had  shown  the  mechanism  of  the 

*  "  Zeitschrif t  f.  ration.  Med.,"  1869,  Bd.  xxxvi — quoted  by  Nuttall  in  his 
"Blood  Immunity  and  Relationships." 

f  "Zur  Lehre  von  der  Bluttransfusion,"  Leipzig,  1875. 

j  "  Transactions  of  the  College  of  Physicians  of  Philadelphia,"  1897,  p.  105. 

§  "Journal  of  Exp.  Med.,"  1901-1905,  vi,  p.  277. 

||  "Archiv.  f.  Exp.  Path,  and  Pharmak.,"  xxv,  pp.  in  and  135. 
**  "Compt.  rendu  de  la  Soc.  de  Biol.  de  Paris,"  1898,  p.  129. 
ft" Berliner  klin.  Wochenschrift.,"  1898. 
it  "Jour,  de  la  R.  Acad.  d.  Med.  de  Torino,"  1898,  No.  8. 
§§  "Ann.  de  PInst.  Pasteur,"  1898,  xn,  688. 
III!  "Berliner  klin.  Wochenschrift,"  1899. 


134  Immunity 

hemolytic  action.  From  this  time  on  the  literature  of  hemolysis 
rapidly  grew  and  the  subject  assumed  a  more  and  more  important 
place  in  the  domain  of  chemico-physiological  research. 

The  technic  of  hemolysis  is  comparatively  simple,  and  it  is  intended 
in  this  chapter  to  do  no  more  than  offer  the  student  a  simple  method 
of  performing  experiments  which  he  can  modify  to  suit  his  own 
purposes. 

For  the  study  of  hemolysis  and  hemo-agglutination  it  is  necessary  to  prepare 
a  5  per  cent,  suspension  of  the  blood-corpuscles  in  an  iso tonic  salt  (NaCl)  solu- 
tion. To  do  this  the  blood  of  the  animal  is  permitted  to  flow  into  a  sterile  tube 
and  is  immediately  stirred  with  a  small  stick  or  a  platinum  wire  until  completely 
defibrinated.  Some  salt  solution  (0.85-0.9  per  cent.)  is  then  added  and  the 
mixture  shaken.  It  is  then  placed  in  a  sterile  centrifuge  tube  and  rotated  until 
the  corpuscles  are  packed  in  a  mass  at  the  bottom.  The  supernatant  fluid  is 
poured  off,  replaced  by  an  equal  volume  of  salt  solution,  and  shaken  until  the 
corpuscles  are  again  thoroughly  distributed.  It  is  then  again  centrifugated  and 
the  fluid  again  poured  off,  after  which  95  parts  (by  volume  as  compared  with 
the  corpuscular  mass)  of  the  salt  solution  are  added  and  the  fluid  thoroughly 
shaken  to  distribute  the  corpuscles.  This  slightly  greenish-red  fluid  is  the  5  per 
cent,  solution  of  corpuscles.  It  is,  of  course,  not  permanent,  and  easily  spoils 
if  bacteria  enter.  It  also  gradually  deteriorates  through  changes  in  the  cor- 
puscles, so  that  it  is  not  usually  useful  after  the  third  day,  even  when  kept  on  ice. 

The  hemolytic  substance  to  be  investigated  must  be  isotonic  with  the  corpuscles 
and  therefore  must  be  dissolved  in,  or  diluted  with,  the  same  salt  solution  as 
that  used  for  making  the  corpuscular  suspension.  Neglect  to  observe  this  re- 
quirement may  lead  to  error  by  diminishing  the  tonicity  of  the  solution  and 
inducing  spontaneous  or  hypotonic  disintegration  of  the  corpuscles. 

To  secure  a  specifically  hemolytic  serum  one  injects  an  animal — say  a  rabbit 
or  guinea-pig — with  increasing  doses  of  the  washed  blood  corpuscles  of  the  animal 
for  whose  corpuscles  the  serum  is  to  be  made  hemolytic,  the  doses  being  given 
intraperitoneally  about  six  times,  at  intervals  of  a  week.  The  animal  is  then  bled, 
the  blood  permitted  to  coagulate,  the  serum  separated  and  filtered,  if  necessary. 

The  contact  of  the  corpuscles  and  the  hemolytic  substance  is  best  conducted 
in  small  test-tubes  holding  about  2  cc.  of  the  mixed  fluids.  It  is  usually  best  to 
work  with  a  constant  volume  of  the  blood-corpuscle  suspension  and  varying 
quantities  or  concentrations  of  the  hemolytic  substances.  Two  observations 
are  to  be  made,  one  after  thirty  minutes'  sojourn  in  the  thermostat  at  3 7°C., 
the  other  after  twenty-four  hours  in  the  ice-box,  both  observations  being  made  on 
the  same  series  of  tubes.  Hemolysis  is  shown  by  the  appearance  of  a  beautiful 
clear  red  color  of  the  formerly  cloudy  greenish  suspension.  One  must  notice  the 
difference  between  partial  and  complete  hemolysis,  different  additions  of  the 
hemolytic  substance  being  required  for  these  results. 

Cytolysis. — The  phenomena  of  hemolysis  corresponds  to  those 
by  which  many  other  cells,  vegetable  and  animal,  are  destroyed  and 
dissolved  through  the  activity  of  immunity  products.  Delezene* 
first  produced  a  leukolytic  or  leukocyte-destroying  serum  by  in- 
jecting animals  with  the  leukocytes  of  a  heterologous  species; 
Metalnikofl,t  by  injecting  the  spermatozoa  of  one  animal  into 
another  of  another  species,  produced  a  spermatoxic  or  spermalytic 
serum;  von  Dlingern,t  a  serum  capable  of  dissolving  the  ciliated 
epithelium  scraped  from  the  trachea  of  an  ox  by  injecting  the 
dissociated  epithelial  cells  into  an  animal,  Delezene§  found  that 

*"Compt.  rendu  de  1'Acad.  de  Sciences  de  Paris,"  1900. 
"Ann.  de  1'Inst.  Pasteur,"  1899. 
^Munchener  med.  Wochenschrift,"  1899. 

§  "Compt.  rendu  de  PAcad.  de  Sciences  de  Paris,"  1900,  cxxx,  pp.  938  and 
1488. 


Bacteriolysis  135 

by  injecting  an  animal  with  the  dissociated  liver  cells  of  a  heter- 
ologous  animal,  a  hepatolytic  serum  could  be  produced. 

The  technic  of  these  investigators  is  not  difficult.  It  is,  however,  first  neces- 
sary to  prepare  a  homogeneous  tissue  pulp  for  injection  into  the  animal  that  is 
to  furnish  immune  serum.  For  this  purpose  it  is  necessary  to  grind  the  tissues, 
when  solid,  in  some  kind  of  mill,  one  of  the  best  forms  of  apparatus  being  that 
of  Latapie.*  After  the  pulp  is  made,  it  is  diluted  to  a  convenient  extent  with 
physiological  salt  solution  and  then  injected  into  the  experiment  animal  in  the 
same  manner  as  is  the  blood  for  making  the  hemolytic  serum.  After  animal  has 
received  a  number  of  injections  made  at  intervals  of  a  few  days  and  is  thought 
to  be  "immunized"  it  is  bled  and  the  serum  separated.  The  remaining  steps 
in  the  experiment  do  not  differ  essentially  from  those  of  hemolytic  experiments. 
The  tissue  suspension,  having  about  the  same  concentration  as  the  5  per  cent. 
NaCl  suspensions  of  the  corpuscles,  is  used  as  the  constant  quantity  and  the 
immune  serum  used  as  the  variable  quantity.  The  tissue  suspension  or  antigen, 
the  immune  serum  or  amboceptor,  and  the  complement  in  normal  guinea-pig 
serum  are  brought  into  contact  in  small  test-tubes,  kept  for  twenty-four  hours  in 
the  refrigerator,  and  the  amount  of  solution  gauged  by  the  naked  eye  supple- 
mented by  microscopical  examination  of  the  tissue  elements. 


Fig.  28. — Latapie's    instrument    for   preparing    tissue    pulp. 


Bacteriolysis. — The  first  observations  upon  bacteriolysis  were 
made  in  1874  by  Traube  and  Gscheidel,|  who  found  that  freshly 
drawn  blood  was  destructive  to  bacteria.  The  matter  was  pur- 
sued by  numerous  subsequent  investigators  and  was  explained  by 
Buchner  as  depending  upon  alexines.  PfeifferJ  described  the 
peculiar  reaction  known  as  "Pfeiffer's  phenomenon."  Ehrlich  and 
Morgenroth§  and  Bordet||  described  the  mechanism  of  cytolysis, 
explaining  the  "Pfeiffer  phenomenon "  and  paving  the  way  for 
future  experiments. 

Direct  destruction  of  bacteria  by  blood-serum  and  body  juices 
is  rare,  and  occurs  only  when  the  serum  contains  appropriate 

*  "Ann.  de  PInst.  Pasteur,"  1902,  xvi,  p.  947. 
t"Jahresb.  der  schles.  Ges.  f.  vaterl.  Kultur,"  1874. 
I  "Deutsche  med.  Wochenschrift,"  1896,  No.  7. 
~  "Berliner  klin.  Wochenschrift,"  1899. 
"Ann.  de  ITnst.  Pasteur,"  1898,  xii. 


136  Immunity 

quantities  of  both  factors  involved — i.e.,  amboceptor  and  com- 
plement. For  the  usual  bacteriolytic  investigations  it  is,  therefore, 
necessary  to  consider  three  factors :  i ,  The  bacteria  to  be  destroyed ; 
2,  the  serum  furnishing  the  complement;  and  3,  the  serum  furnish- 
ing the  immune  body. 

Technic. — i.  The  bacteria  to  be  destroyed  should  be  prepared  in  the  form  of  a 
homogeneous  suspension  in  physiological  salt  solution,  similar  to  that  employed 
for  making  the  agglutination  tests  (q.  i>.).  It  is  best  to  use  the  surface  growths 
from  agar-agar,  well  -rubbed  upon  the  side  of  a  test-tube  containing  the  fluid, 
which  is  permitted  to  contact  with  the  mass  from  time  to  time  by  inclining  the 
tube  so  that  the  fluid  is  able  to  carry  away  the  bacteria  as  they  are  distributed. 

If  quantitative  estimations  are  to  be  made,  the  number  of  bacteria  in  the  sus- 
pension must  be  known  or  at  least  a  standard  quantity  must  be  employed, 
as  the  destructive  process  is  a  chemical  one,  in  which  the  destructive  agents  are 
themselves  used  up. 

2.  The  serum  furnishing  the  complement  is  a  normal  serum — that  is,  the 
serum  from  a  healthy  animal  that  has  undergone  no  manipulation.     The  guinea- 
pig  is  the  animal  preferred. 

3.  The  serum  containing  the  amboceptor  or  the  immune  body  is  obtained 
from  an  animal  that  has  been  given  a  high  degree  of  immunization  against  the 
bacterium  to  be  destroyed  or  dissolved.     The  complement  contained  in  this 
serum  should  be  destroyed  by  heating  for  a  short  time  to  55°C. 

These  three  having  been  prepared,  an  appropriate  quantity  of  the  bacterial 
suspension  is  placed  in  a  small  test-tube,  and  an  appropriate  quantity  of  the 
diluted  normal  serum  added.  To  this  mixture  of  two  constants,  varying  quanti- 
ties of  the  immune  serum  are  added  and  the  tube  stood  away  for  twenty-four 
hours  on  ice.  In  almost  every  case  it  will  be  found  that  the  immune  serum  con- 
tains a  great  quantity  of  agglutinating  substance,  so  that  the  bacteria  all  fall  to 
the  bottom  in  a  short  time.  This  is  independent  of  bacteriolysis.  The  bacterial 
destruction  is  gauged  by  the  disappearance  of  the  bacteria  or  by  their  failure  to 
grow  when  transplanted  to  appropriate  culture  media. 

By  making  the  bacterial  suspension  and  complementary  serum  constant  quan- 
tities (taking  care  that  not  too  many  bacteria  be  present),  one  is  able  to  estimate 
the  value  of  the  immune  serum.  By  using  the  bacterial  suspension  and  a  heated 
immune  serum  (containing  no  complement)  as  constants  and  varying  the  addi- 
tion of  complementary  serum,  one  can  estimate  the  respective  values  of  several 
complementary  serums.  By  using  both  serums  as  constant  factors  and  varying 
the  number  of  bacteria,  one  can  determine  the  exact  bacteriolytic  value  of  the 
mixture.  By  taking  out  and  planting  drops  from  time  to  time  the  rapidity  of 
bacteriolysis  can  be  determined,  and  by  plating  out  the  drops  and  counting  the 
colonies  one  may  arrive  at  percentages  of  destruction  and  express  the  bacteriolytic 
process  in  the  form  of  a  curve. 

THE   DEVIATION    OF   THE    COMPLEMENT,  OR  THE  "NEISSER-WECHSBERG 

PHENOMENON" 

A  peculiar  phenomenon  has  been  observed  and  studied  by  Neisser 
and  Wechsberg.*  When  an  animal  whose  blood-serum  is  nor- 
mally possessed  of  a  high  degree  of  germicidal  power  is  immunized 
by  repeated  injections  of  a  bacterial  antigen,  its  serum  when  ex- 
amined by  the  usual  methods  fails  to  show  the  usual  increase  in 
the  specific  bactericidal  action  toward  that  particular  organism, 
though  it  retains  its  general  bacteria-destroying  power.  If,  however, 
the  serum  be  greatly  diluted,  its  action  is  changed,  so  that  it  loses 
its  general  bacteria-destroying  power  and  develops  marked  increase 
in  the  specific  destructive  action  upon  the  particular  bacteria  used 

*  "Munch,  med.  Wochenschrift,"  April  30,  1901,  XLVIII,  No.  13,  p.  697. 


The  Deviation  of  the  Complement 


in  the  experiment.  Neisser  and  Wechsberg  attribute  the  peculiar 
reaction  to  the  fact  that  there  being  more  amboceptors  than  com- 
plements in  the  serum,  some  of  the  former  satisfy  their  combining 
affinities  by  attaching  themselves  to  the  bacteria,  some  by  attach- 
ing themselves  to  the  complement,  instead  of  forming  combinations 
of  all  three.  If  under  these  circumstances  the  serum  containing 


A1 


Fig.  29. — Diagram  illustrating  the  Neisser- Wechsberg  phenomenon  of  "de- 
viation of  complement."  In  A1  the  three  black  units  (c)  represent  the  quantity 
of  complement  necessary  for  the  dissolution  of  a  bacterium,  and  the  three  white 
units  (6)  the  intermediate  bodies  or  amboceptors  through  which  they  may  act. 
A2  shows  these  properly  proportioned  units  properly  combined  and  anchored  to 
the  bacterial  cell  which  will  be  destroyed.  If  an  excess  of  amboceptor  units  be 
present,  as  is  suggested  in  B1,  the  resulting  combinations  and  the  consequent 
results  may  vary  according  to  the  differing  combining  affinities.  Thus,  B 2  shows 
an  unchanged  affinity,  i.e.,  only  those  amboceptors  unite  with  bacterial  cells 
that  are  charged  with  complement.  C2  shows  equal  affinity  of  the  amboceptors 
for  complement  and  for  the  bacterial  cell,  so  that  charged  or  uncharged  units 
attach  themselves  to  the  cell,  diminishing  the  complementary  action.  D2  shows 
the  possible  result  when  the  affinity  of  the  amboceptor  for  the  bacterial  cell  is 
diminished  after  charging  with  complement,  so  that  though  the  complement  and 
amboceptor  combine,  there  can  be  no  destruction  of  the  bacterium.  Thus,  excess 
of  the  amboceptor  units  may  "deviate  the  complement"  and  prevent  its  action. 

the  amboceptors  is  diluted  until  their  number  becomes  approximately 
equal  to  the  number  of  complements  introduced,  any  deviation 
resulting  from  inequality  of  the  combining  affinities  becomes  im- 
probable. Bordet  and  Gay,*  however,  have  performed  experiments 
tending  to  show  that  these  elements  do  not  really  unite,  thus  seem- 
*  "Ann.  de  PInst.  Pasteur,"  June  25,  1906,  xx,  No.  6,  pp.  267-498. 


138 


Immunity 


ing  to  controvert  the  theory  of  Neisser  and  Wechsberg,  and  Bolton* 
has  shown  that  normal  serum  may  kill  relatively  more  bacteria  when 
diluted  than  when  undiluted. 

THERAPEUTIC  USES  OF  BACTERIOLYTIC  SERUMS 

It  was  at  first  hoped  that  some  of  these  serums  and  especially 
the  bacteriolytic  serums  would  have  a  wide  therapeutic  application 
in  cases  in  which  non-toxicogenic  bacteria  were 
invading  the  body,  but  experiment  and  experi- 
ence have  shown  that  the  laws  governing  their 
action  greatly  limit  their  application,  and  that 
their  effects,  when  not  beneficial,  are  bound  to 
be  harmful.  The  difficulty  lies  in  the  fact  that 
when  we  manufacture  such  serums  we  prepare 
only  the  immune  body,  there  being  no  increase 
of  the  complement. 

To  introduce  this  by  itself  does  the  patient 
no  good,  because  in  most  cases  the  existing  in- 
fection has  brought  about  the  formation  of  as 
much  or  more  "immune  body"  than  can  be  util- 
ized by  the  complement.  To  give  injections  of 
active  bodies  that  cannot  be  utilized  is  shown  by 
Comus  and  Gleyf  and  Kosself  to  be  followed 
by  the  formation  of  antibodies— in  this  case 
"anti-immune  bodies" — by  which  their  effect 
is  neutralized.  Should  anti-immune  bodies  be 
formed  by  this  meddlesome  medication,  the 
state  of  the  infected  animal  would  be  worse 
than  before,  because  it  would  now  be  preparing 
that  which  by  neutralizing  the  combining  affini- 
ties of  its  own  immune  bodies,  would  prevent 
them  from  combining  with  the  elements  to  be 
destroyed  and  so  activating  the  complements. 
No  satisfactory  method  of  experimentally  increasing  the  comple- 
ment has  been  devised.  If,  as  Metschnikoff  supposes,  the  comple- 
ment is  microcytase  derived  from  disintegrated  leukocytes,  aseptic 
suppurations  with  active  phagolysis  should  result  in  marked  increase 
of  the  complement.  As  a  matter  of  fact,  this  does  take  place,  but 
the  increase  is  so  slight  that  the  serum  is  not  practically  valuable. 
Therapeutic  serums  whose  practical  application  is  based  upon 
their  cytolytic  activity  must,  of  necessity,  contain  both  the  essential 
factors  involved  in  cytolysis,  and  should  contain  them  in  such  pro- 
portions that,  regardless  of  other  elements  in  the  blood,  they  can 
exercise  their  combining  and  dissolving  functions. 

*  "The  Bacteriolytic  Power  of  the  Blood-serum  of  Hogs,"  Bull.  No.  950!  the 
Bureau  of  Animal  Industry,  U.  S.  Dept.  of  Agriculture. 

f  "Compte  rendu  de  1'Acad.  de  Sciences  de  Paris,"  Jan.  i,  1898,  126. 
j"Berl.  klin.  Woch.,"  1898,  S.  152. 


Fig.  30. — Schemat- 
ic representation  of 
the  interfering  ac- 
tion of  anti-ambo- 
ceptors,  and  anti- 
complements.  A, 
Anti-a  mboceptor 
action:  c,  Comple- 
ment; am,  ambo- 
ceptor;  aa,  anti-am- 
boceptor  preventing 
the  amboceptor  from 
connecting  with  the 
cell.  B:  c,  Com- 
plement; ac,  anti- 
complement  pre- 
venting the  comple- 
ment from  connect- 
ing with  the  ambo- 
ceptor, am. 


Complement  Fixation  139 

We  are  unable  experimentally  to  accomplish  these  prerequisites, 
therefore  are  not  in  the  position  to  accurately  apply  bacteriolytic 
serums  in  practice. 

COMPLEMENT  FIXATION 

In  1901  Bordet,  while  investigating  the  nature  of  the  comple- 
mentary substance,  made  a  discovery  that  has  now  become  of  great 
importance,  that  is,  the  " Bordet-Gengou  phenomenon,"  or,  as  it 
is  now  known,  the  " fixation  of  the  complement."  His  method  of 
procedure  was  as  follows:  Blood-corpuscles  were  sensitized  with 
appropriate  amboceptors  and  then  treated  with  freshly-drawn  nor- 
mal serum.  Hemolysis  resulted.  If  now  he  added  to  the  mixture 
some  sensitized  blood-corpuscles  of  a  different  species,  they  did  not 
hemolyze.  Clearly,  the  complement  had  been  used  up  in  the  first 
hemolysis. 

He  next  found  that  if,  instead  of  employing  blood-corpuscles  for 
the  first  test,  he  used  sensitized  bacteria — i.e.,  bacteria  treated  with 
an  immune  serum  containing  the  amboceptors  appropriate  for  effect- 
ing their  solution — the  complement  would  similarly  be  used  up, 
"  fixed,"  so  that  when  he  subsequently  added  sensitized  red  blood- 
corpuscles  there  was  no  hemolysis. 

This  reaction  was  naturally  quantitative,  the  result  as  described 
depending  upon  the  fact  that  no  more  complement  (normal  serum) 
was  used  in  the  original  hemolysis  or  bacteriolysis  than  was  necessary 
and  so  none  left  "unfixed"  to  effect  the  lysis  or  solution  of  the  second 
factor  introduced. 

Bordet  interpreted  his  results  as  indicating  that  there  was  only 
one  complementary  or  solvent  substance,  and  though  Ehrlich  sub- 
sequently published  what  he  looked  upon  as  proofs  to  the  contrary, 
the  opinion  of  Bordet  prevails. 

In  addition,  however,  Bordet's  experiments  have  been  of  practical 
use.  As  affording  a  means  of  quantitative  experimentation  they 
have  enabled  investigators  to  measure  the  quantity  of  complement 
in  normal  bloods  and  in  immunized  bloods,  and  so  led  to  the  discovery 
that  for  each  kind  of  animal  and  for  each  individual  animal  the 
complement  is  subject  to  very  little  variation.  In  the  course  of 
some  three  years  they  were  followed  by  the  investigations  of  Neisser 
and  Sachs  upon  antigens,  and  made  to  subserve  the  useful  purpose 
of  recognizing  and  differentiating  antigenic  substances.  Thus, 
when  a  certain  antibody  and  its  complement  are  combined  they  can 
only  attach  themselves  to  the  particular  specific  antigen  by  which 
the  antibody  has  been  developed.  But,  what  is  still  more  important, 
they  have  led  to  the  invention  of  methods  by  which  the  presence  of 
specific  amboceptors  may  be  determined  where  they  are  suspected, 
and  so  have  made  possible  means  of  arriving  at  a  correct  diagnosis 
in  certain  obscure  cases  of  disease  in  man. 

The  most  important  of  these  measures  is  the  Wassermann  reac- 


140  Immunity 

tion  for  the  diagnosis  of  syphilis  (q.v.).  By  careful  perusal  of  the 
chapter  upon  the  method  of  performing  the  Wassermann  reaction 
the  student  will  learn  the  general  details  of  the  technic  of  complement 
fixation,  and  can  modify  them  to  correspond  to  the  requirements 
of  other  cases  in  which  complement  fixation  is  to  be  studied. 

DEFENSIVE  FERMENTS 

Defensive  ferments  are  enzymic  substances  that  make  their 
appearance  in  the  body  juices  in  a  short  time  after  any  unusual 
protein  substance  is  intentionally  or  accidentally  thrown  into  the 
blood.  They  were  discovered  by  Abderhalden*  who  found  that 
when  substances  capable  of  digestive  transformation  in  the  animal 
economy,  by  any  means  obtain  access  to  the  blood,  ferments  capable 
of  effecting  such  transformations  also  quickly  appear  in  the  blood 
in  increased  quantity,  effect  the  transformation  and  then  quickly 
disappear.  The  appearance  and  disappearance  of  the  enzymes  is 
supposed  to  depend  upon  "  mobilization  "  of  defensive  ferments,  of 
which  the  body  presumably  has  reserve  supplies.  The  most  common 
source  of  supply  is  supposed  to  be  the  leukocytes. 

The  Abderhalden  Reaction. — The  subject  was  first  investigated 
with  reference  to  the  presence  of  a  proteolytic  ferment  in  the  blood 
of  pregnant  woman,  whose  office  was  the  defense  of  the  mother 
against  the  syncytial  and  chorionic  cells  of  the  offspring  which  with 
their  products  may  occasionally  get  into  the  circulation. 

If  such  a  ferment  were  present  in  the  blood,  it  ought  to  be  demon- 
strably  capable  of  effecting  transformations  in  the  sub-stratum  by 
whose  presence  it  has  been  called  forth.  To  determine  it,  therefore, 
it  should  only  be  necessary  to  apply  the  blood  serum  to  the  sub- 
stratum for  a  brief  time,  and  then  determine  by  sufficiently  delicate 
tests  that  some  transformation  has  been  effected.  For  the  latter 
Abderhalden  has  made  use  of  two  separate  tests: 

The  first  of  these  is  rarely  employed,  the  second  is  now  regularly 
employed. 

I.  The  Optical    Test. — This   depends  upon  the  fact  that  in  the 
transformation  of  protein  substances,  aminoacids  may  be  formed, 
some  of  which  are  optically  active.     The  contact  of  the  enzymic 
serum  and  the  appropriate  sub-stratum  is  permitted  to  take  place, 
then  after  the  appropriate  length  of  time,  the  polariscope  is  employed 
to  determine  whether  rotation   differences  obtain  because  of  the 
presence  of  transformation  products. 

II.  The  Dialysis  Test. — This  test  not  requiring  apparatus  or  skill 
of  unusual  or  special  kind,  has  met  with  greater  favor  and  is  now  in 
daily  use.     Its  first  employment  was  for  the  demonstration  of  the 
presence,  in  the  blood,  of  an  enzyme  that  would  transform  placenta! 
tissue.    As  no  such  enzyme  appeared  in  the  blood  except  placental 

*  "Schiitzfermente  des  tierische  Organismus,"  Berlin,  1912;  Berlin,  1913. 


Defensive  Ferments  141 

tissue  was  in  the  body,  it  became  a  test  for  the  determination  of 
the  existence  of  pregnancy.  The  method  required  but  little  in  the 
way  of  special  apparatus  or  reagents.  The  chief  requirements 
being  small  "dialyzing  shells"  or  thimbles,  which  are  made  by 
Schleichter  and  Schull,  and  are  commercially  known  as  No.  5790. 
They  are  procurable  through  importing  agents  dealing  in  laboratory 
apparatus.  These  shells  must  be  tested  before  using,  and  it  is  best 
to  test  a  large  number  at  the  same  time.  Each  must  be  impervious 
to  albumen,  but  readily  permeable  to  peptones,  aminoacids  and 
other  cleavage  products  of  protein  digestion. 

The  shells  or  " thimbles"  are  tested  thus  by  Kolmer:*— 

They  are  first  soaked  in  sterile  distilled  water  for  half  an  hour  or  more,  until 
they  are  softened.  Each  then  receives  about  2.5  cc.  of  a  5  per  cent,  solution  of 
egg-albumen  in  distilled  water,  thoroughly  mixed  and  free  from  flakes  or  shreds. 
In  filling  the  shell,  care  should  be  exercised  that  none  of  the  albumen  solution  by 
any  chance  falls  upon  the  outside.  The  shell  is  then  picked  up  with  forceps  and 
transferred  to  a  short  tube  containing  about  20  cc.  of  sterile  distilled  water. 
This  tube  should  be  so  wide  that  the  column  of  water  is  not  so  deep  as  the  shell  is 
high,  and  not  so  broad  that  the  shell  is  in  danger  of  oversetting.  As  bacteria 
may  not  have  been  successfully  excluded  and  by  multiplying  may  cause  proteo- 
lytic  cleavage  of  the  albumen,  it  is  well  to  cover  the  fluid  in  the  thimble  and  that 
in  the  tube  outside  of  it,  with  a  thin  layer  of  toluol.  The  outer  tube  is  plugged  or 
corked,  and  the  whole  is  stood  in  the  incubating  oven  where  it  is  kept  at  37°C. 
for  sixteen  to  eighteen  hours.  At  the  end  of  this  time,  10  cc.  of  the  water  in  the 
outer  tube  is  removed  by  a  pipette,  and  tested  by  the  biuret  reaction  to  determine 
whether  any  albumen  has  penetrated  the  thimble.  For  this  purpose  the  fluid, 
in  a  test-tube,  receives  2.5  cc.  of  a  33  per  cent,  solution  of  sodium  hydroxid  and  is 
shaken  gently.  One  cubic  centimeter  of  a  0.2  per  cent,  cupric  sulphate  solution 
is  permitted  to  trickle  down  the  side  of  the  tube  and  overlie  the  contents.  If  a 
delicate  violet  is  produced  at  the  line  of  junction  of  the  two  liquids,  albumen  has 
escaped  from  the  thimble  into  the  water  outside.  Under  such  circumstances  the 
thimble  is,  of  course,  useless  and  should  be  thrown  away.  ^  If  there  is  any  uncer- 
tainty about  the  reaction,  the  tube  can  be  stood  away  for  eight  hours  or  so  longer 
(twenty-four  hours  in  all)  and  the  remaining  water  subjected  to  the  ninhydrin 
test  (see  below). 

The  good  shells  or  thimbles  are  next  to  be  tested  for  permeability  to  peptones. 
Before  this  they  should  be  carefully  washed  in  running  water  and  boiled  for 
thirty  seconds. 

A  i  per  cent,  solution  of  Hochst  "silk  peptone"  is  made  in  distilled  water,  and 
of  it  2.5  cc.  is  pipetted  into  each  thimble  to  be  tested,  taking  care,  as  before,  that 
none  of  the  solution  by  accident  drops  on  the  outside  of  the  shell.  The  shell  is 
now  placed  in  the  20  cc.  of  sterile  distilled  water  in  the  wide  tube  such  as  was 
used  before,  covered  with  toluol  and  stood  in  the  incubator  at  37°C.  After 
twenty-four  hours,  a  pipette  is  thrust  through  the  toluol  and  10  cc.  of  the  water 
taken  up.  The  finger  being  held  over  the  top  of  the  pipette,  the  tube  is  wiped 
outside  with  care,  so  as  to  get  off  any  toluol,  and  the  fluid  then  delivered  into  a 
test-tube.  Here  it  receives  0.2  cc.  of  a  i  per  cent,  solution  of  ninhydrin,  and  is 
boiled  for  exactly  one  minute.  If  the  peptone  has  dialyzed,  a  deep  blue  color 
develops  after  standing  for  a  short  time.  The  thimble  that  permits  no  transfu- 
sion of  peptone  is  worthless  and  should  be  thrown  away. 

The  good  thimbles  are  now  again  thoroughly  washed  in  running  water  for  a 
minute,  or  so,  and  are  then  transferred  to  a  vessel  of  sterile  distilled  water  con- 
taining chloroform  to  saturation  and  covered  with  toluol. 

In  making  the  Abderhalden  test  it  is  imperative  that  the  glass- 
ware used  should  be  chemically  clean,  that  the  reagents  be  pure, 
that  the  preparations  be  kept  sterile  and  that  the  thimbles  and  sub- 
strata should  be  handled  with  forceps,  not  with  the  fingers. 

*  "Infection,  Immunity  and  Specific  Therapy."    Phila.,  1915;  p.  253. 


14  2  Immunity 

To  make  the  test  for  pregnancy  known  as  the  "Abderhalden  reac- 
tion," the  foundation  of  all  the  other  tests  of  the  protective  or  defen- 
sive ferments,  it  is  necessary  to  prepare  a  substratum  upon  which 
the  enzyme  in  the  blood  may  act. 

To  do  this  one  obtains  a  healthy  placenta,  removes  the  blood  clots,  cord  and 
membranes,  and  washes  it  in  running  water.  When  it  is  clean  on  the  outside, 
it  is  cut  into  small  pieces — i  cm.  cubes — which  are  placed  upon  a  towel  or  on  a 
wire  sieve  and  washed  in  running  water.  The  purpose  of  the  washing  is  to  remove 
every  trace  of  blood  serum  and  of  blood  pigment.  From  time  to  time  the  bits 
of  tissue  are  moved  about  and  squeezed  by  the  fingers,  and  occasionally  they  are 
crushed  together  in  a  towel.  The  process  is  completed  when  the  tissue  has  be- 
come perfectly  white  in  color.  It  now  receives  100  times  its  weight  of  distilled 
water  (i  gram-i  cc.),  to  which  are  added  five  drops  of  glacial  acetic  acid  per 
1000  cc.,  and  is  boiled  for  ten  minutes.  The  fluid  is  then  thrown  away,  the  tissue 
fragments  are  caught  in  a  sieve  or  cloth,  more  distilled  water  added,  this  time 
without  the  acetic  acid,  and  it  is  boiled  again.  This  is  repeated  for  six  times. 
After  the  sixth  boiling,  some  of  the  water  is  transferred  to  a  tube  and  tested  for 
proteins  with  ninhydrin.  If  the  faintest  blue  color  develops  upon  boiling,  the 
process  of  washing  the  tissue  by  boiling  it  with  clean  water,  must  be  repeated 
again  and  again  until  the  ninhydrin  produces  no  discoloration  after  boiling  for  a 
minute,  and  standing  for  one-half  hour.  The  tissue  is  then  caught  on  a  cloth, 
finally  looked  over  for  any  objectionable  components,  and  transferred  to  a  jar  of 
sterile  distilled  water  saturated  with  chloroform  and  covered  with  toluol. 

The  blood  of  the  patient  is  obtained  with  a  Keidel  tube  or  with  a 
sterile  syringe  from  which  latter  it  is  at  once  transferred  to  a  sterile 
test-tube.  When  the  blood  has  firmly  coagulated,  the  expressed 
serum  is  removed  by  a  sterile  pipette  to  a  sterile  centrifuge  tube  and 
any  cells  it  may  still  contain  are  thrown  out  by  centrifugation. 

The  technic  of  the  test  is  more  simple  than  the  preparation  and 
preliminary  tests  it  entailed.  The  glassware  being  chemically  clean 
and  sterile,  the  thimbles  all  tested  and  sterile,  and  the  substratum 
(placental  tissue)  ready  one  proceeds  as  follows: 

A  fragment  of  the  placental  tissue  is  removed  from  the  container  with  sterile 
forceps  and  blotted  with  sterile  filter  or  blotting  paper  to  absorb  the  toluol  and 
chloroform.  It  is  then  placed  upon  a  sterile  filter  paper  and  weighed;  about 
0.5  gram  should  be  placed  in  each  of  two  thimbles.  1.5  cc.  of  the  serum  to  be 
tested  is  cautiously  pipetted  into  one  thimble;  1.5  cc.  of  sterile  distilled  water 
into  the  other.  Each  is  then  transferred  with  forceps  to  a  large  tube  containing 
20  cc.  of  sterile  distilled  water,  and  the  surface  of  each  fluid  is  covered  with 
toluol.  The  tubes  are  now  stood  in  the  thermostat  at  37°C.  for  twenty-four 
hours,  at  the  end  of  which  time  a  sample  of  the  fluid  in  each  outer  tube  is  tested 
by  boiling  for  one  minute  with  ninhydrin  (0.2  cc.  of  a  i  per  cent,  solution,  to  locc. 
of  the  fluid).  The  reaction  is  not  read  for  thirty  minutes  after  boiling.  If  the 
conditions  are  all  favorable,  i.e.,  the  serum  used  be  from  a  pregnant  woman,  the 
tissue  used  as  substratum  beplacenta,theenzymeintheserumactsuponthesub- 
stratum  and  transforms  its  albumins  to  peptones  and  amino-acids;  if  the  trans- 
fusion is  perfect  in  both  thimbles,  and  neither  thimble  leaks  (this  has,  of  course, 
been  previously  tested  and  security  can  be  counted  upon  now)  the  fluid  surround- 
ing the  thimble  containing  the  serum  should  give  a  bright  blue  color  or  positive 
reaction,  and  that  surrounding  the  thimble  containing  the  water  no  color  or  a 
negative  reaction. 

By  the  test  we  are  then  able  to  determine,  the  substratum  being 
known,  whether  the  serum  contains  an  enzyme  capable  of  acting 
upon  or  transforming  it ;  or  the  enzymic  character  of  the  serum  being 
known,  it  may  be  possible  to  tell  something  about  the  substratum. 


Defensive  Ferments  143 

The  general  consensus  of  opinion  is  in  favor  of  this  reaction  as  being 
a  useful  adjunct  in  making  the  diagnosis  of  pregnancy.  But  its 
applicability  may  not  be  limited  to  the  diagnosis  of  pregnancy  for 
Freund  and  Abderhalden,*  Frank  and  Heimanf  and  many  others 
have  used  it  as  an  adjunct  in  the  diagnosis  of  cancer,  and  various 
other  investigators  have  shown  that  modifications  of  the  method 
makes  it  applicable  for  purposes  of  diagnosis  or  investigation  of  other 
conditions  in  which  defensive  enzymes  may  be  present  in  the  blood. 
For  each  of  these  investigations  the  specific  substratum  must  be  pre- 
pared, and  in  making  each  test,  the  application  of  the  enzyme-con- 
taining serum  to  the  sterile  and  appropriate  substratum  must  be  made 
in  the  tested  thimbles  with  the  precautions  given  above. 

The  method  is  not  exclusively  adapted  for  investigation  of  proteo- 
lytic  enzymes  in  the  serum,  but  to  diastatic  and  lipolytic  ferments 
as  well  and  Abderhalden  has  shown  that  it  has  uses  in  these  fields. 
How  much  importance  attaches  to  the  enzymes  thus  mobilized  in 
the  blood  in  the  conditions  comprehended  in  the  studies  of  immunity 
is  as  yet  uncertain.  That  there  is  some  bearing  of  the  one  upon  the 
other  cannot  be  doubted.  The  Abderhalden  reactions  seem  to  be 
less  specific  than  the  immunity  reactions  and  appear  more  as  reac- 
tions en  gros,  while  the  immunity  reactions  previously  studied 
were  reactions  en  detail,  but  it  may  well  be  that  this  apparent  differ- 
ence depends  upon  the  newness  of  the  former  reactions  and  the 
crudity  of  the  methods  employed  as  contrasted  with  the  more 
elaborate  study  of  the  latter  and  the  more  delicate  methods  used. 

*  Munch,  med.  Wochenschrift,  1913,  xiv,  763. 
t  Berl.  klin.  Wochenschrift,  1913,  L,  No.  14. 


CHAPTER  V 
METHODS  OF  OBSERVING  MICRO-ORGANISMS 

IT  is  of  the  utmost  importance  to  examine  micro-organisms 
alive,  and  as  nearly  as  possible  in  their  normal  environment,  then  to 
supplement  this  examination  by  the  study  of  dead  and  stained 
specimens. 

The  study  of  the  living  organism  has  the  advantage  of  showing 
its  true  shape,  size,  grouping,  motility,  reproduction,  and  natural 
history.  It  has  the  disadvantage  of  being  somewhat  difficult  because 
of  its  small  size  and  transparency. 

So  long  as  bacteria  were  observed  only  in  the  natural  condition, 
however,  it  was  impossible  to  find  them  in  the  tissues  of  diseased 
animals,  and  it  was  not  until  Weigert  suggested  the  use  of  the  anilin 
dyes  for  coloring  them  that  their  demonstration  was  made  easy 
and  their  relationship  to  pathologic  conditions  established. 

The  beauty  and  clearness  of  stained  specimens,  and  the  ease 
with  which  they  can  be  observed,  have  led  to  some  serious  errors 
on  the  part  of  students,  who  often  fail  to  realize  the  unnatural  con- 
dition of  the  stained  bacteria  they  observe.  It  only  needs  a  moment's 
consideration  to  show  how  disturbed  must  be  the  structure  of 
an  organism  after  it  has  been  dried,  fixed,  boiled,  or  steamed, 
passed  through  several  chemic  reagents,  dehydrated  and  impreg- 
nated with  stains,  etc.,  to  suggest  how  totally  unnatural  its  appear- 
ance may  become. 

It  is,  therefore,  necessary  to  examine  every  organism,  under 
study,  in  the  living  condition,  and  to  control  all  the  appearances 
of  the  stained  specimen  by  comparison. 

I.  THE  STUDY  OF  LIVING  BACTERIA 

The  simplest  method  of  observing  live  bacteria  is  to  take  a 
drop  of  liquid  containing  them,  place  it  upon  a  slide,  put  on  a 
cover,  and  examine. 

While  this  method  is  simple,  it  cannot  be  recommended,  as 
evaporation  at  the  edges  causes  currents  of  liquid  to  flow  to  and 
fro  beneath  the  cover,  carrying  the  bacteria  with  them  and  making 
it  almost  impossible  to  determine  whether  the  organisms  under  ex- 
amination are  motile  or  not.  Should  it  be  desirable  that  such  a 
specimen  be  kept  for  a  time,  so  much  evaporation  takes  place  that 
in  the  course  of  an  hour  or  two  it  has  changed  too  much  to  be  of 
further  use. 

144 


The  Study  of  Living  Bacteria 


145 


The  best  way  to  examine  living  micro-organisms  is  in  what  is 
called  the  hanging  drop.  A  hollow-ground  slide  is  used,  and  with 
the  aid  of  a  small  camel's-hair  pencil  a  ring  of  vaselin  is  drawn  on  the 
slide  about,  not  in,  the  concavity.  A  drop  of  the  material  to  be 
examined  is  placed  in  the  center  of  a  large  clean  cover-glass  and 
then  placed  upon  the  slide  so  that  the  drop  hangs  in,  but  does  not 
touch,  the  glass.  The  micro-organisms  are  thus  hermetically 
sealed  in  an  air  chamber,  and  appear  under  almost  the  same  con- 
ditions as  in  the  culture.  Such  a  specimen  may  be  kept  and  ex- 
amined from  day  to  day,  the  bacteria  continuing  to  live  until  the 
oxygen  or  nutriment  is  exhausted.  By  means  of  a  special  ap- 
paratus in  which  the  microscope  is  placed,  the  growing  bacteria 
may  be  watched  at  any  temperature,  and  exact  observations  made. 

The  hanging  drop  should  always  be  examined  at  the  edge,  as  the 
center  is  too  thick. 

In  such  a  specimen  it  is  possible  to  determine  the  shape,  size, 


Fig.  31. — The  "hanging  drop"  seen  from  above  and  in  profile. 

grouping,  division,  sporulation,  and  motility  of  the  organism  under 
observation. 

Care  should  be  exercised  to  use  a  rather  small  drop,  especially  for 
the  detection  of  motility,  as  a  large  one  vibrates  and  masks  the 
motility  of  the  sluggish  forms. 

When  the  bacteria  to  be  observed  are  in  solid  or  semi-solid  culture, 
a  small  quantity  of  the  culture  should  be  mixed  in  a  drop  of  sterile 
bouillon  or  other  fluid. 

For  observing  the  growth  of  bacteria  where  it  is  desirable  to 
prevent  movement,  Hill*  has  invented  an  ingenious  device  which  he 
calls  the  "hanging  block."  His  directions  for  preparing  it  are  as 
follows: 

"Pour  melted  nutrient  agar  into  a  Petri  dish  to  the  depth  of  about  one-eighth 
or  one-quarter  inch.  Cool  this  agar,  and  cut  from  it  a  block  about  one-quarter 
inch  to  one-third  inch  square  and  of  the  thickness  of  the  agar  layer  in  the  dish. 
This  block  has  a  smooth  upper  and  under  surface.  Place  it,  under  side  down,  on 
a  slide  and  protect  it  from  dust.  Prepare  an  emulsion,  in  sterile  water,  of  the 
organism  to  be  examined  if  it  has  been  grown  on  a  solid  medium,  or  use  a  broth 
culture;  spread  the  emulsion  or  broth  upon  the  upper  surface  of  the  block  as 

*"  Journal  of  Medical  Research,"  March,  1902,  vol.  vn,  No.  2;  new  series, 
vol.  ii. 


146  Methods  of  Observing  Micro-organisms 

if  making  an  ordinary  cover-slip  preparation.  Place  the  slide  and  block  in  a 
37°C.  incubator  for  five  to  ten  minutes  to  dry  slightly.  Then  lay  a  clean  sterile 
cover-slip  on  the  inoculated  surface  of  the  block  in  close  contact  with  it,  usually 
avoiding  air-bubbles.  Remove  the  slide  from  the  lower  surface  of  the  block  and 
invert  the  cover-slip  so  that  the  agar  block  is  uppermost.  With  a  platinum 
loop  run  a  drop  or  two  of  melted  agar  along  each  side  of  the  agar  block,  to  fill 
the  angles  between  the  sides  of  the  block  and  the  cover-slip.  This  seal  hardens 
at  once,  preventing  slipping  of  the  block.  Place  the  preparation  in  the  incubator 
again  for  five  or  ten  minutes  to  dry  the  agar-agar  seal.  Invert  this  preparation 
over  a  moist  chamber  and  seal  the  cover-slip  in  place  with  white  wax  or  paraffin. 
Vaselin  softens  too  readily  at  37°C.,  allowing  shifting  of  the  cover-slip.  The 
preparation  may  then  be  examined  at  leisure." 

With  this  means  of  examining  the  growing  cultures,  Hill  has  ac- 
quired interesting  knowledge  of  the  fission  and  budding  of  Bacillus 
diphtheriae. 

If  the  specimens  to  be  examined  must  be  kept  for  some  time  at 
an  elevated  temperature,  some  such  apparatus  as  that  of  Nuttall 
will  be  found  useful. 


H.  STAINING  BACTERIA 

In  the  early  days  of  bacteriology  efforts  were  made  to  facilitate 
the  observation  of  bacteria  by  the  use  of  nuclear  dyes.  Both  carmin 
and  hematoxylin  tinge  the  nuclei  of  the  bacteria  a  little,  but  so  un- 
satisfactorily that  since  Weigert  introduced  the  anilin  dyes  for  the 
purpose,  all  other  stains  have  been  abandoned.  The  affinity  be- 
tween the  bacteria  and  the  anilin  dyes  is  peculiar,  and  in  certain 
cases  can  be  used  for  the  differentiation  of  species. 

The  best  anilin  dyes  made  at  the  present  time,  and  those  which 
have  become  the  standard  for  all  bacteriologic  work,  are  made  in 
Germany  by  Dr.  Griibler,  and  in  ordering  stains  the  name  of  this 
manufacturer  should  be  specified. 

Readers  interested  in  the  biochemistry  of  the  subject  will  do  well 
to  refer  to  the  excellent  papers  by  Arnold  Grimme,*  upon  "The 
Important  Methods  of  Staining  Bacteria,  etc.,"  and  Marx,f  upon 
"The  Metachromatic  and  Babes-Ernst  Granules." 
.  In  this  -work  special  methods  for  staining  such  bacteria  as  have 
peculiar  reactions  will  be  given  together  with  the  description  of  the 
particular  organisms,  general  methods  only  being  discussed  in  this 
chapter. 

Preparations  for  General  Examination. — For  bacteriologic  pur- 
poses thin  covers  (No.  i)  are  required,  because  thicker  glasses  may 
interfere  with  the  focussing  of  the  oil-immersion  lenses.  The  cover- 
glasses  must  be  perfectly  clean.  It  is  therefore  best  to  clean  a  large 
quantity  in  advance  of  use  by  immersing  them  first  in  a  strong  mineral 
acid,  then  washing  them  in  water,  then  in  alcohol,  then  in  ether, 
and  finally  keeping  them  in  ether  until  they  are  to  be  used.  Except 
that  it  sometimes  cracks,  bends,  or  fuses  the  edge  of  the  glass,  a 

*  "Centralbl.  f.  Bakt.,"  etc.,  1902,  Bd.  xxxn,  Nos.  2,  3,  4,  and  5. 
f  Ibid.,  1902,  xxxii,  Nos.  10  and  n,  p.  108. 


Simple  Method  of  Staining 


147 


more  convenient  method  is  to  wipe  the  glasses  as  clean  as  possible 
with  a  soft  cotton  cloth,  seize  them  with  fine-pointed  forceps,  and 
pass  them  repeatedly  through  a  small  Bunsen  flame  until  it  becomes 
greenish-yellow.  The  hot  glass  must  then  be  slowly  elevated 
above  the  flame,  so  as  to  allow  it  to  anneal.  This  manceuver 
removes  the  organic  matter  by  combustion.  It  is  not  expedient 
to  use  covers  twice  for  bac- 
teriologic  work,  though  if 
well  cleansed  by  immer- 
sion in  acid  and  washing, 
they  may  subsequently 
be  employed  for  ordinary 
microscopic  objects. 

The  fragility  of  the 
covers  and  their  likelihood 
to  be  broken  or  dropped 
at  the  critical  moment, 
make  most  workers  pre- 
fer to  stain  directly  upon 
the  slide.  The  slide  should 
be  thoroughly  cleaned, 
and  if  the  material  to  be 
examined  is  spread  near 
one  end,  the  other  may 
serve  as  a  convenient  han- 
dle. The  slide  is  also  to 
be  preferred  if  a  number 
of  examinations  are  to  be 
made  simultaneously  or 
for  comparison,  as  it  is 
large  enough  to  contain  a 


Fig.  32. — Apparatus  for  keeping  objects  under 
microscopic  examination  at  constant  tempera- 


Simple  Method  of  Stain- 
ing.— The  material  to  be 
examined  must  be  spread 

*  ,  -        «  .  _  JLJ.XJ.VxJL  WvJV.W^y.1.^         V     .\t 

in   the   thinnest   possible    tures  (Nuttall). 
layer  upon  the  surface  of 

the  perfectly  clean  cover-glass  or  slide  and  dried.  The  most  conveni- 
ent method  of  spreading  is  to  place  a  minute  drop  on  the  glass  with 
a  platinum  loop,  and  then  spread  it  evenly  over  the  glass  with  the  flat 
wire.  Should  it  be  stained  at  once  it  would  all  wash  off,  so  it  must 
next  be  fixed  to  the  glass  by  being  passed  three  times  through  aflame, 
experience  having  shown  that  when  drawn  through  the  flame  three 
times  the  desired  effect  is  usually  accomplished.  The  Germans 
recommend  that  a  Bunsen  burner  or  a  large  alcohol  lamp  be  used, 
that  the  arm  describe  a  circle  a  foot  in  diameter,  each  revolution 
occupying  a  second  of  time,  and  the  glass  being  made  to  pass  through 


148  Methods  of  Observing  Micro-organisms 

the  flame  from  apex  to  base  three  times.  This  is  supposed  to  be 
exactly  the  requisite  amount  of  heating.  The  rule  is  a  good  one  for 
the  inexperienced. 

Inequality  in  the  size  of  various  flames  may  make  it  desirable 
to  have  a  more  accurate  rule.  Novy*  suggests  that  as  soon  as  it  is 
found  that  the  glass  is  so  hot  that  it  can  no  longer  be  held  against  the 
finger  it  is  sufficiently  heated  for  fixing. 

After  fixing,  the  preparation  is  ready  for  the  stain.  Every  labora- 
tory should  be  provided  with  "stock  solutions,"  which  are  saturated 
solutions  of  the  ordinary  dyes.  For  preparing  them  Woodf  gives 
the  following  parts  per  100  as  being  sufficiently  accurate: 

Alcoholic  solutions  (96  per  cent,  alcohol)  Aqueous  solutions  (distilled   water) 

Fuchsin 3.0  grams. 

Gentian  violet 4.8       "  Gentian  violet 1.5  grams. 

Methylene-blue 7.0       "  Methylene-blue 6.7       " 

("O  per  cent,  alcohol) 

Scharlach  R 3.2       " 

Soudan  III 0.2       " 

(50  per  cent,  alcohol) 
Thionin 0.6       "          Thionin 1.2       " 

Of  these  it  is  well  to  have  fuchsin,  gentian  violet,  and  methylene- 
blue  always  made  up.  The  stock  solutions  will  not  stain,  but  form 
the  basis  of  the  staining  solutions.  For  ordinary  staining  an  aqueous 
solution  is  employed.  A  small  bottle  is  nearly  filled  with  distilled 
water,  and  the  stock  solution  added,  drop  by  drop,  until  the  color 
becomes  just  sufficiently  intense  to  prevent  the  ready  recognition 
of  objects  through  it.  For  exact  work  it  is  probably  best  to  give 
these  stains  a  standard  composition,  using  5  cc.  of  the  saturated 
alcoholic  solution  to  95  cc.  of  water.  Such  a  watery  solution  pos- 
sesses the  power  of  readily  penetrating  the  dried  cytoplasm  of  the 
bacterium. 

Cover-glasses  are  apt  to  slip  from  the  fingers  and  spill  the  stain, 
so  when  using  them  it  is  well  to  be  provided  with  special  forceps 
which  hold  the  glass  in  a  firm  grip  and  allow  of  all  manipula- 
tions without  danger  of  soiling  the  fingers  or  clothes.  The  ordi- 
nary sharp-pointed  forceps  are  unfit  for  the  purpose,  as  capillary 
attraction  draws  the  stain  between  the  blades  and  makes  certain 
the  soiling  of  the  fingers.  In  using  the  special  forceps  the  glass  should 
not  be  caught  at  the  edge,  but  a  short  distance  from  it,  as  shown  in 
the  cut.  This  altogether  prevents  capillary  attraction  between  the 
blades.  When  the  material  is  spread  upon  the  slide  no  forceps  are 
needed,  and  the  method  correspondingly  simplified.  Sufficient  stain 
is  allowed  to  run  from  a  pipet  upon  the  smear  to  flood  it,  but  not 
overflow,  and  is  allowed  to  remain  for  a  moment  or  two,  after  which 
it  is  thoroughly  washed  off  with  water.  The  smear  upon  a  slide  is 
then  dried  and  examined  at  once,  a  drop  of  oil  of  cedar  being  placed 

*  "Laboratory  Work  in  Bacteriology,"  1899. 

f  "Chemical  and  Microscopical  Diagnosis,"  N.  Y.,  1905,  D.  Appleton  &  Co., 
p.  683. 


Staining  Bacteria  in  Tissues  149 

directly  upon  the  smear,  and  no  cover-glass  used.  If  the  staining 
has  been  done  upon  a  cover-glass,  it  can  be  mounted  upon  a  slide 
with  a  drop  of  water  between,  and  then  examined,  though  this  is 
less  satisfactory  than  examination  after  drying  it  and  mounting  it 
in  Canada  balsam. 

Sometimes  the  material  to  be  examined  is  solid  or  too  thick  to 
spread  upon  the  glass  conveniently.  Under  such  circumstances  a 
drop  of  distilled  water  or  bouillon  can  be  added  and  a  minute  portion 
of  the  material  mixed  in  it  and  spread  upon  the  glass. 

When  the  bacteria  are  contained  in  urine  or  other  non-albuminous 
fluid,  so  that  the  heat  used  for  fixing  has  nothing  to  coagulate  and 
fix  the  organisms  to  the  glass,  a  drop  of  Meyer's  glycerin-albumen 
can  be  added  with  advantage,  though  the  precaution  must  be  taken 
to  see  that  this  mixture  contains  no  bacteria  to  cause  confusion  with 
those  in  the  material  to  be  studied. 

The  entire  process  is,  in  brief:  (i)  Spread  the  material  upon  the 
glass;  (2)  dry — do  not  heat;  (3)  pass  three  times  through  the  flame; 
(4)  stain — one  minute;  (5)  wash  thoroughly  in  water;  (6)  dry;  (7) 
mount  in  Canada  balsam. 


Fig.  33. — Stewart's  cover-glass  forceps. 

To   Observe  Bacteria  in  Sections  of  Tissue. — Hardening. — It 

not  infrequently  happens  that  the  bacteria  to  be  examined  are  scat- 
tered among  or  inclosed  in  the  cells  of  tissues.  The  demonstration 
then  becomes  a  matter  of  difficulty,  and  the  method  employed  must 
be  modified  according  to  the  particular  kind  of  organism.  The 
success  of  the  method  will  depend  upon  the  good  preservation  of  the 
tissue  to  be  studied.  As  bacteria  disintegrate  rapidly  in  dead  tissue, 
the  specimen  for  examination  should  be  secured  as  fresh  as  possible, 
cut  into  small  fragments,  and  immersed  in  absolute  alcohol  from  six 
to  twenty-four  hours,  to  kill  and  fix  the  cells  and  bacteria.  The 
blocks  are  then  removed  from  the  absolute  alcohol  and  kept  in  80 
to  90  per  cent,  alcohol,  which  does  not  shrink  the  tissue.  Solutions 
of  bichlorid  of  mercury*  may  also  be  used  and  are  particularly  useful 
when  the  bacteria  are  to  be  studied  in  relation  to  the  cells  of  the 
tissues. 

*  Zenker's  fluid: 

Bichromate  of  potassium 2.5  grams 

Sulphate  of  sodium i .  o 

Bichlorid  of  mercury. 5.0 

Water 100.0 

At  the  time  of  using  add  5  grams  of  glacial  acetic  acid.  Permit  the  specimens 
to  remain  in  the  solution  for  a  few  hours  only,  then  wash  for  twenty-four  hours  in 
running  water  and  transfer  to  80  per  cent,  alcohol. 


150  Methods  of  Observing  Micro-organisms 

Tissues  preserved  in  95  per  cent,  alcohol,  Miiller's  fluid,  4  per 
cent,  formaldehyd,  and  other  ordinary  solutions  rarely  show  the 
bacteria  well. 

Embedding. — The  ordinary  methods  of  embedding  suffice.  The 
simpler  of  these  are  as  follows: 

/.  Celloidin  (Schering). — The  solutions  of  celloidin  are  made  in 
equal  parts  of  absolute  alcohol  and  ether  and  should  have  the  thick- 
ness of  oil  or  molasses.  From  the  hardening  reagent  (if  other  than 
absolute  alcohol)  pass  the  blocks  of  tissue  through: 

Ninety-five  per  cent,  alcohol,  twelve  to  twenty-four  hours; 
Absolute  alcohol,  six  to  twelve  hours; 

Thin  celloidin  (consistence  of  oil),  twelve  to  twenty-four  hours; 
Thick  celloidin  (consistence  of  molasses),  six  to  twelve  hours. 

Place  upon  a  block  of  vulcanite  or  hard  wood,  allow  the  ether 
to  evaporate  until  the  block  can  be  overturned  without  dislodging 
the  specimen;  then  place  in  80  per  cent,  alcohol  until  ready  to  cut. 
The  knife  must  be  kept  flooded  with  alcohol  while  cutting. 

Celloidin  is  soluble  in  absolute  alcohol,  ether,  and  oil  of  cloves, 
so  that  the  staining  of  the  sections  must  be  accomplished  without 
the  use  of  these  reagents  if  possible. 

Celloidin  sections  can  be  fastened  to  the  slide,  if  desired,  by 
firmly  pressing  filter  paper  upon  them  and  rubbing  hard,  then 
allowing  a  little  vapor  of  ether  to  run  upon  them. 

//.  Paraffin. — Pure  paraffin  having  a  melting-point  of  about 
52°C.  is  used.  The  hardened  blocks  of  tissue  are  passed  through: 

Ninety-five  per  cent,  alcohol,  twelve  to  twenty- four  hours; 
Absolute  alcohol,  six  to  twelve  hours; 
Chloroform,  benzole,  or  xylol,  four  hours; 

A  saturated  solution  of  paraffin  in  one  of  the  above  reagents,  four  to  eight 
hours. 

The  block  is  then  placed  in  melted  paraffin  in  an  oven  or  paraffin 
water-bath,  at  5o°-55°C.,  until  the  volatile  reagent  is  all  evaporated, 
and  the  tissue  impregnated  with  paraffin  (four  to  twelve  hours), 
and  finally  embedded  in  freshly  melted  paraffin  in  any  convenient 
mold.  In  cutting,  the  knife  must  be  perfectly  dry. 

The  cut  paraffin  sections  can  be  placed  upon  the  surface  of 
slightly  warmed  water  to  flatten  out  the  wrinkles,  and  then  floated 
upon  a  clean  slide  upon  which  a  film  of  Meyer's  glycerin-albumen 
(equal  parts  of  glycerin  and  white  of  egg  thoroughly  beaten  up  and 
filtered,  and  preserved  with  a  crystal  of  thymol)  has  been  spread. 
After  drying,  the  slides  are  placed  in  the  paraffin  oven  for  an  hour 
at  60° C.,  so  that  the  albumen  coagulates  and  fixes  the  sections  to 
the  glass. 

When  sections  so  spread  and  fixed  upon  the  slide  are  to  be  stained, 
the  paraffin  must  first  be  dissolved  in  chloroform,  benzole,  xylol, 
oil  of  turpentine,  etc.,  which  in  turn  must  be  removed  with  95  per 
cent,  alcohol.  The  further  staining,  by  whatever  method  desired,  is 
accomplished  by  dropping  the  reagents  upon  the  slide. 


Staining  151 

III.  Glycerin-gelatin. — As  the  penetration  of  the  tissue  by 
celloidin  is  attended  with  deterioration  in  the  staining  qualities  of 
the  tubercle  bacillus,  it  has  been  recommended  by  Kolle*  that  the 
tissue  be  saturated  with  a  mixture  of  glycerin,  i  part;  gelatin,  2 
parts;  and  water,  3  parts;  cemented  to  a  cork  or  block  of  wood, 
hardened  in  absolute  alcohol,  and  cut  as  usual  for  celloidin  with  a 
knife  wet  with  alcohol. 

Staining. — Simple  Method. — For  ordinary  work  the  following 
simple  method  can  be  recommended:  After  the  sections  are  cut 
and  cemented  to  the  slide,  the  paraffin  and  celloidin  should  be  re- 
moved by  appropriate  solvents.  The  sections  are  immersed  in  the 
ordinary  aqueous  solution  of  the  anilin  stain  and  allowed  to  re- 
main about  five  minutes,  next 
washed  in  water  for  several  min- 
utes, then  decolorized  in  0.5  to 
i  per  cent,  acetic  acid  solution. 
The  acid  removes  the  stain  from 
the  tissues,  but  ultimately  from 
the  bacteria  as  well,  so  that  one 
must  watch  carefully,  and  so  soon 
as  the  color  has  almost  disap- 
peared from  the  sections,  they 
must  be  removed  and  transferred 
to  absolute  alcohol.  At  this  point 
the  process  may  be  interrupted  to 
allow  the  tissue  elements  to  be 
countercolored  with  alum-carmin 
or  any  stain  not  requiring  acid 
for  differentiation,  after  which  the  sections  are  dehydrated  in 
absolute  alcohol,  cleared  in  xylol,  and  mounted  in  Canada  balsam. 

The  greater  number  of  applications  can  be  made  by  simply 
dropping  the  reagents  upon  the  slide  while  held  in  the  fingers. 
Where  exposure  to  the  reagents  is  to  be  prolonged,  the  Coplin  jar 
or  some  more  capacious  device  must  be  employed. 

Pfeiffer's  Method. — The  sections  are  stained  for  one-half  hour  in 
diluted  ZiehPs  carbol-fuchsin  (q.v.),  then  transferred  to  absolute 
alcohol  made  feebly  acid  with  acetic  acid.  The  sections  must 
be  carefully  watched,  and  so  soon  as  the  original,  almost  black- 
red  color  gives  place  to  a  red-violet  color  they  are  removed  to  xylol, 
to  be  cleared  preparatory  to  mounting  in  balsam. 

Loffler's  Method. — Certain  bacteria  that  do  not  permit  ready 
penetration  by  the  dye  require  some  more  intense  stain.  One  of 
the  best  of  these  is  LofHer's  alkaline  methylene-blue: 

Saturated  alcoholic  solution  of  methylene-blue. 30 

i  :  10,000  aqueous  solution  of  caustic  potash 100 


CROSS-SECTION 
SHOWING  SLIDES 
IN  POSITION. 


Fig.  34. — Coplin's  staining  jar. 


*Fliigge's  "Die  Mikroorganismen,"  vol.  i,  page  534. 


152  Methods  of  Observing  Micro-organisms 

The  cut  sections  of  tissue  are  stained  for  a  few  minutes  and 
then  differentiated  in  a  i  per  cent,  solution  of  hydrochloric  acid  for 
a  few  seconds,  after  which  they  are  dehydrated  in  alcohol,  cleared  in 
xylol,  and  mounted  in  balsam. 

Some  bacteria,  such  as  the  typhoid  fever  bacillus,  decolorize 
readily  so  that  the  use  of  acid  should  be  avoided,  washing  in  water 
or  alcohol  being  sufficient. 

Gram's  Method  of  Staining  Bacteria  in  Tissue. — Gram  was 
the  fortunate  discoverer  of  a  method  of  impregnating  bacteria 
with  an  insoluble  color.  It  will  be  seen  at  a  glance  that  this  is  a 
marked  improvement  on  the  methods  given  above,  as  the  stained 
tissue  can  be  washed  thoroughly  in  either  water  or  alcohol  until  its 
cells  are  colorless,  without  fear  that  the  bacteria  will  be  decolorized. 
The  details  of  the  method  are  as  follows:  The  section  is  stained 
from  five  to  ten  minutes  in  a  solution  of  a  basic  anilin  dye,  pure 
anilin  (anilin  oil)  and  water.  This  solution,  first  devised  by  Ehrlich, 
is  known  as  Ehrlich's  solution.  The  ordinary  method  of  preparing 
it  is  to  mix  the  following: 

Pure  anilin 4 

Saturated  alcoholic  solution  of  gentian  violet n 

Water 100 

Instead  of  gentian  violet,  methyl  violet,  Victoria  blue,  or  any 
pararosanilin  dye  will  answer.  The  rosanilin  dyes,  such  as  fuchsin, 
methylene-blue,  vesuvin,  etc.,  will  not  react  with  iodin,  and  so 
cannot  be  used  for  the  purpose.  The  anilin-oil  solutions  do  not  keep 
well;  in  fact,  seldom  longer  than  six  to  eight  weeks,  sometimes  not 
more  than  two  or  three;  therefore  it  is  best  to  prepare  but  a  small 
quantity  by  pouring  about  i  cc.  of  pure  anilin  into  a  test-tube, 
filling  the  tube  about  one-half  with  distilled  water,  shaking  well, 
then  filtering  as  much  as  is  desired  into  a  small  dish.  To  this  the 
saturated  alcoholic  solution  of  the  dye  is  added  until  the  surface 
becomes  distinctly  metallic  in  appearance. 

Friedlander  recommends  that  the  section  remain  from  fifteen  to 
thirty  minutes  in  warm  stain,  and  in  many  cases  the  prolonged 
process  gives  better  results. 

From  the  stain  the  section  is  given  a  rather  hasty  washing  in 
water,  and  then  immersed  from  two  to  three  minutes  in  Gram's 
solution  (a  dilute  Lugol's  solution): 

Iodin  crystals i 

Potassium  iodid 2 

Water 300 

The  specimen  while  in  the  Gram  solution  turns  a  dark  blackish- 
brown  color,  but  when  removed  and  carefully  washed  in  95  per 
cent,  alcohol  again  becomes  blue.  The  washing  in  95  per  cent, 
alcohol  is  continued  until  no  more  color  is  given  off  and  the  tissue 
assumes'its  original  color.  If  it  is  simply  desired  to  find  the  bacteria, 
the  section  can  be  dehydrated  in  absolute  alcohol  for  a  moment, 


Staining  153 

cleared  in  xylol,  and  mounted  in  Canada  balsam.  If  it  is  necessary 
to  study  the  relation  of  the  bacteria  to  the  tissue  elements,  a  nuclear 
stain,  such  as  alum-carmin  or  Bismarck  brown,  may  be  previously 
or  subsequently  used.  Should  a  nuclear  stain  requiring  acid  for 
its  differentiation  be  desirable,  the  process  of  staining  must  precede 
the  Gram  stain,  so  that  the  acid  shall  not  act  upon  the  stained 
bacteria. 

Gram's  method  rests  upon  the  fact  that  the  combination  of  bacterial 
substance,  anilin  dye,  and  the  iodids  forms  a  compound  insoluble  in 
alcohol. 

The  process  described  may  be  summed  up  as  follows: 

Stain  in  Ehrlich's  anilin-water  gentian  violet  five  to  thirty  minutes; 

Wash  in  water; 

Immerse  two  to  three  minutes  in  Gram's  solution; 

Wash  in  95  per  cent,  alcohol  until  no  more  color  comes  out; 

Dehydrate  in  absolute  alcohol; 

Clear  in  xylol; 

Mount  in  Canada  balsam. 

No  matter  how  carefully  the  method  is  performed,  an  unsightly 
precipitate  is  sometimes  deposited  upon  the  tissue,  obscuring  both 
its  cells  and  contained  bacteria.  Muir  and  Ritchie  obviate  this 
(i)  by  making  the  staining  solution  with  i  120  aqueous  solution  of 
carbolic  acid  instead  of  the  saturated  anilin  solution,  and  (2)  by 
clearing  the  tissue  with  oil  of  cloves  after  dehydration  with  alcohol. 
The  oil  of  cloves,  however,  is  itself  a  powerful  decolorant  and  must 
be  washed  out  in  xylol  before  the  section  is  mounted  in  Canada 
balsam. 

Gram's  method  is  also  employed  to  aid  in  differentiating  similar 
species  of  bacteria  in  culture.  A  thin  layer  of  a  suspension  of  the 
bacteria  to  be  examined  is  spread  upon  a  slide  or  cover-glass,  dried, 
and  fixed;  then  flooded  with  the  anilin-oil  gentian  violet  or  other 
staining  solution.  The  solution  is  kept  warm  by  holding  the  glass 
flooded  with  the  stain  over  a  small  flame.  The  process  of  staining 
is  continued  from  two  to  five  minutes.  If  the  heating  causes  the 
'stain  to  evaporate,  more  of  it  must  be  added  so  that  it  does  not 
dry  and  incrust  the  glass. 

The  stain  is  poured  off,  and  replaced  by  Gram's  solution,  which 
is  allowed  to  remain  from  one-half  to  two  minutes,  and  gently 
agitated. 

The  smear  is  next  washed  in  95  per  cent,  alcohol  until  the  blue 
color  is  wholly  or  almost  lost,  after  which  it  can  be  counterstained 
with  pyronin,  eosin,  Bismarck  brown,  vesuvin,  etc.,  washed,  dried, 
and  mounted  in  Canada  balsam.  Given  briefly,  the  method  is: 

Stain  with  Ehrlich's  solution  two  to  five  minutes; 

Gram's  solution  for  one-half  to  two  minutes; 

Wash  in  95  per  cent,  alcohol  until  decolorized; 

Counterstain  if  desired;  wash  off  the  counterstain  with  water; 

Dry; 

Mount  in  Canada  balsam. 


154  Methods  of  Observing  Micro-organisms 

Nicolle*  suggests  the  following  modification  of  the  technic: 

(a)  For  Cover-glass  Specimens: 

1.  Stain  for  one  to  five  minutes  in  a  warm  solution  made  as  follows:  10  cc. 

of  saturated  alcoholic  solution  of  gentian  violet,  100  cc.  of  a  i  per  cent, 
aqueous  solution  of  carbolic  acid. 

2.  Immerse  from  four  to  six  seconds  in  the  iodine-iodide  of  potassium  solu- 

tion. 

3.  Decolorize  in  a  mixture  of  3  parts  of  absolute  alcohol  and  i  part  of  acetone. 

4.  Counterstain  if  desired. 

(b)  For  Sections: 

1.  Stain  the  nuclear  elements  of  the  tissue  with  carmine.     For  this  Nicolle 

prefers  Orth's  carmine  solution  (5  parts  of  Orth's  carmine  with  i  part 
of  95  per  cent,  alcohol). 

2.  Stain  in  the  carbol-gentian  violet,  as  indicated  above. 

3.  Immerse  for  four  to  six  seconds  in  the  iodine-iodide  of  potassium  solu- 

tion. 

4.  Differentiate  with  absolute  alcohol  containing  0.33  per  cent,  (by  volume) 

of  acetone. 

5.  Treat  with  95  per  cent,  alcohol  containing  some  picric  acid  until  the 

tissue  is  greenish  yellow  (one  to  five  seconds). 

6.  Dehydrate  with  absolute  alcohol. 

7.  Clear  with  xylol  or  other  appropriate  reagent. 

8.  Mount  in  balsam. 

The  Gram-Weigert  Stain  can  be  employed  with  beautiful  results 
for  staining  many  micro-organisms.  It  differs  from  the  Gram 
method  in  that  anilin  oil  instead  of  alcohol  is  used  for  decolorizing. 
To  secure  the  most  brilliant  results  it  is  best  first  to  stain  the  tissue 
with  alum,  borax,  or  lithium  carmin,  and  then — 

1.  Stain  in  Ehrlich's  anilin-oil-water  gentian  violet,  five  to  twenty  minutes; 

2.  Wash  off  excess  with  normal  salt  solution; 

3.  Immerse  in  dilute  iodin  solution  (iodin  i,  iodid  of  potassium  2,  water  100) 

for  one  minute; 

4.  Drain  off  the  fluid  and  blot  the  section  spread  out  upon  the  slide,  with 

absorbent  paper; 

5.  Decolorize  with  a  mixture  of  equal  parts  of  anilin  and  xylol; 

6.  Wash  out  the  anilin  with  pure  xylol. 

7.  Mount  in  xylol  balsam. 

Gram's  method  does  not  stain  all  bacteria,  hence  can  be  used  to 
aid  in  the  differentiation  of  species: 

Gram-negative  Gram-positive 

Bacillus  anthracis  symptomatici;  Bacillus  aerogenes  capsulatus; 

Bacillus  coli  (whole  group);  Bacillus  anthracis; 

Bacillus  ducreyi;  Bacillus  botulinus; 

Bacillus  dysenteriae  Bacillus  diphtherias; 

Bacillus  icteroides;  Bacillus  subtilis  (whole  group); 

Bacillus  influenzas;  Bacillus  tetani; 

Bacillus  mallei;  Bacillus  tuberculosis  (whole  acid- 
Bacillus  cedematis  maligni;  fast  group); 

Bacillus  pestis  bubonica;  Diplococcus  pneumonia?; 

Bacillus  pneumonias  (Friedlander);  Micrococcus  tetragenus; 

*  "Ann.  de  PInst.  Pasteur,"  1895,  rx. 


Staining  155 

Gram-negative  Gram-positive 

Bacillus  proteus  vulgaris;  Staphylococcus  pyogenes  albus; 

Bacillus  pyocyaneus;  Staphylococcus  pyogenes  aureus; 

Bacillus  rhinoscleromatis;  Streptococcus  pyogenes. 

Bacillus  suipestifer; 
Bacillus  suisepticus; 
Bacillus  typhosus  (whole  group) ; 
Diplococcus  intracellularis  meningitidis; 
Micrococcus  catarrhalis; 
Micrococcus  gonorrhoea?  (Neisser) 
Micrococcus  melitensis; 
Spirillum  cholerse  asiaticse; 
Spirillum  cholera?  gallinarum; 
Spirillum  cholerse  nostras; 
Spirillum  metschnikovi; 
Spirillum  tyrogenum; 
Spirochaete  duttoni; 
Spirochaete  obermeieri; 
Spirochaete  refringens; 
Treponema  pallidum; 
Treponema  pertenue. 

Eosin  and  Methylene-blue  (Mallory)  make  a  beautiful  contrast 
tissue  stain  for  routine  work,  and  also  demonstrate  the  presence 
of  most  bacteria.  The  success  of  the  method  seems  to  depend  largely 
upon  the  quality  of  the  reagents  used  and  a  careful  study  of  their 
effects.  Hardening  in  Zenker's  fluid  is  highly  recommended  as  a 
preliminary.  The  details  as  given  by  Mallory  are  as  follows: 

1.  Stain  paraffin  sections  in  a  5  to  10  per  cent,  aqueous  solution  of  eosin 

from  five  to  twenty  minutes  or  longer; 

2.  Wash  in  water  to  get  rid  of  the  excess  of  eosin; 

3.  Stain  in  Unna's  alkaline  methylene-blue  solution  (methylene-blue  i,  car- 

bonate of  potassium  i,  water  100),  diluted  i  :  10  with  water,  from  one- 
half  to  one  hour,  or  use  a  stronger  solution  and  stain  for  a  few  minutes 
only; 

4.  Wash  in  water. 

5.  Differentiate  and  dehydrate  in  95  per  cent,  alcohol,  followed  by  absolute 

alcohol  until  the  pink  color  returns  in  the  section; 

6.  Clear  with  xylol; 

7.  Mount  in  xylol  balsam. 

The  nuclei  and  micro-organisms  will  be  colored  blue,  the  cyto- 
plasm, etc.,  red. 

Zieler*  recommends  for  the  staining  of  the  typhoid,  glanders  and 
other  difficultly  stainable  bacteria,  the  following  method  of  demon- 
stration in  the  tissues: 

1.  Fix  and  harden  in  Miiller-formol  solution. 
Paraffin  imbedding. 

f  Orcein  D o.i 

2.  Staining  overnight  in  j  Officinal  sulphuric  acid 2 . 

(  70  per  cent,  alcohol 100 . 

3.  Washing  in  70  per  cent,  alcohol  for  a  short  time  to  remove  the  excess  of 

orcein. 

4.  Washing  in  water. 

5.  Staining  in  polychrome  methylene-blue  ten  minutes  to  two  hours. 

6.  Washing  in  distilled  water. 

7.  Thorough  differentiation  in  glycerin-ether  i  :  2-5  water  until  the  tissues 

become  pale  blue. 
*  "Centralbl.  f.  allg.  Path.  u.  path.  Anat."     Bd.  xiv,  No.  14,  p.  561. 


156  Methods  of  Observing  Micro-organisms 

8.  Washing  in  distilled  water. 

9.  Seventy  per  cent,  alcohol. 

10.  Absolute  alcohol. 

11.  Xylol. 

12.  Balsam. 

Glanders  bacilli  appear  dark  violet  on  a  colorless  background; 
typhoid  bacilli  intense  dark  red  violet. 

Method  of  Staining  Spores. — It  has  already  been  pointed  out  that 
the  peculiar  quality  of  the  spore  capsules  protects  them  to  a  certain 
extent  from  the  influence  of  stains  and  disinfectants.  On  this  ac- 
count they  are  much  more  difficult  to  color  than  the  adult  bacteria. 
Several  methods  are  recommended,  the  one  generally  employed  being 
as  follows:  Spread  the  thinnest  possible  layer  of  material  upon  a 
cover-glass,  dry,  and  fix.  Have  ready  a  watch-crystalful  of  Ehrlich's 
solution,  preferably  made  of  fuchsin,  and  drop  the  cover-glass, 
prepared  side  down,  upon  the  surface,  where  it  should  float.  Heat 
the  stain  until  it  begins  to  steam,  and  allow  the  specimen  to  remain 
in  the  hot  stain  for  from  five  to  fifteen  minutes.  The  cover  is  then 
transferred  to  a  3  per  cent,  solution  of  hydrochloric  acid  in  absolute 
alcohol  for  about  one  minute.  Abbott  recommends  that  the  cover- 
glass  be  submerged,  prepared  side  up,  in  a  dish  of  this  solution  and 
gently  agitated  for  exactly  one  minute,  removed,  washed  in  water, 
and  counterstained  with  an  aqueous  solution  of  methyl  or  methylene- 
blue. 

In  such  a  specimen  the  spores  should  appear  red,  and  the  adult 
organisms  blue. 

A  good  simple  method  is  to  place  the  prepared  cover-glass  in  a 
test-tube  half  full  of  carbol-f uchsin : 

Fuchsin i 

Alcohol 10 

Five  per  cent,  aqueous  solution  of  phenol  crystals 100 

and  boil  it  for  at  least  fifteen  minutes,  after  which  it  is  decolorized, 
either  with  3  per  cent,  hydrochloric  or  2-5  per  cent,  acetic  acid,  washed 
in  water,  and  counterstained  blue. 

Muir  and  Ritchie*  recommend  that  cover-films  be  prepared  and 
stained  as  for  tubercle  bacilli  (q.v.),  decolorized  with  a  i  per  cent, 
sulphuric  acid  solution  in  water  or  methyl  alcohol,  then  washed  in 
water  and  counterstained  with  a  saturated  aqueous  methylene-blue 
solution  for  half  a  minute,  washed  again  with  water,  dried,  and 
mounted  in  Canada  balsam. 

Abbott's  method  of  staining  spores  is  as  follows: 

1.  Stain  deeply   with   methylene-blue,   heating  repeatedly  until  the  stain 

reaches  the  boiling-point — one  minute. 

2.  Wash  in  water. 

3.  Wash  in  95  per  cent,  alcohol  containing  0.2  to  0.3  per  cent,  of  hydrochloric 

acid. 

4.  Wash  in  water. 

*  "Manual  of  Bacteriology,"  London,  1897. 


Staining  157 

5.  Stain  for  eight  to  ten  seconds  in  anilin-fuchsin  solution. 

6.  Wash  in  water. 

7.  Dry. 

8.  Mount  in  balsam. 

The  spores  are  blue;  the  bacteria,  red. 

Moller*  finds  it  advantageous  to  prepare  the  films,  before  staining, 
by  immersion  in  chloroform  for  two  minutes,  following  this  by 
immersion  in  5  per  cent,  chromic  acid  solution  for  one-half  to  two 
minutes. 

The  exact  technic  is  as  follows: 

1.  Treat  the  spread  with  chloroform  for  two  minutes. 

2.  Wash  with  water. 

3.  Treat  with  5  per  cent,  solution  of  chromic  acid  for  one-half  to  two  minutes. 

4.  Wash  in  water. 

5.  Stain  with  carbol-fuchsin,  slowly  heating  until  the  fluid  boils. 

6.  Decolorize  in  5  per  cent,  aqueous  sulphuric  acid. 

7.  Wash  well  with  water. 

8.  Stain  in  a  i  :  100  aqueous  solution  of  methylene-blue  for  thirty  seconds. 
The  spores  should  be  red  and  the  bacilli  blue. 

Anjeszkyf  recommends  the  following  method  of  staining  spores, 
which  is  said  always  to  give  good  results  even  with  anthrax  bacilli: 
A  cover-glass  is  thinly  spread  with  the  spore-containing  fluid  and 
dried.  While  it  is  drying,  some  0.5  per  cent,  hydrochloric  acid  is 
warmed  in  a  porcelain  dish  over  a  Bunsen  flame  until  it  steams  well 
and  bubbles  begin  to  form.  When  the  solution  is  hot  and  the  smear 
dry,  the  cover-glass  is  dropped  upon  the  fluid,  which  is  allowed  to  act 
upon  the  unfixed  smear  for  three  or  four  minutes.  The  cover  is 
removed,  washed  with  water,  dried,  and  fixed  for  the  first  time,  then 
stained  with  Ziehl's  carbol-fuchsin  solution,  which  is  warmed  twice 
until  fumes  arise.  The  preparation  is  allowed  to  cool,  decolorized 
with  a  4-5  per  cent,  sulphuric  acid  solution,  and  counterstained  for 
a  minute  or  two  with  malachite  green  or  methylene-blue.  The  whole 
procedure  should  not  take  longer  than  eight  or  ten  minutes. 

FioccaJ  suggests  the  following  rapid  method:  "About  20  cc.  of 
a  10  per  cent,  aqueous  solution  of  ammonium  are  poured  into  a 
watch-glass,  and  10  to  20  drops  of  a  saturated  solution  of  gentian 
violet,  fuchsin,  methyl  blue,  or  safranin  added.  The  solution  is 
warmed  until  vapor  begins  to  rise,  then  is  ready  for  use.  A  very 
thinly  spread  cover-glass,  carefully  dried  and  fixed,  is  immersed  for 
three  to  five  minutes  (sometimes  ten  to  twenty  minutes),  washed  in 
water,  washed  momentarily  in  a  20  per  cent,  solution  of  nitric  or 
sulphuric  acid,  washed  again  in  water,  then  counterstained  with  an 
aqueous  solution  of  vesuvin,  chrysoidin,  methyl  blue,  malachite 
green,  or  safranin,  according  to  the  color  of  the  preceding  stain. 
This  whole  process  is  said  to  take  only  from  eight  to  ten  minutes,  and 
to  give  remarkably  clear  and  beautiful  pictures." 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Bd.  x,  p.  273. 

t  Ibid.,  Feb.  27,  1898,  xxm,  No.  8,  p.  329. 

I  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  July  i,  1893,  xiv,  No.  i. 


158  Methods  of  Observing  Micro-organisms 

Method  of  Staining  Flagella. — This  is  more  difficult  than  the 
staining  of  the  bacteria  or  the  spores. 

Loffler's  Method.* — This  is  the  original  and  best  method,  though 
somewhat  cumbersome,  and  hence  rarely  employed  at  the  present 
time.  Three  solutions  are  used: 

(A) — Twenty  per  cent,  aqueous  solution  of  tannic  acid 10 

Cold  saturated  aqueous  solution  of  ferrous  sulphate 5 

Alcoholic  solution  of  fuchsin  or  methyl  violet i 

(B)  One  per  cent,  aqueous  solution  of  caustic  soda. 

(C)  An  aqueous  solution  of  sulphuric  acid  of  such  strength  that  i  cc.  will 
exactly  neutralize  an  equal  quantity  of 'solution  B. 

Some  of  the  culture  to  be  stained  is  mixed  upon  a  cover-glass  with  a  drop  of 
distilled  water  making  a  first  dilution,  which  is  still  too  rich  in  bacteria  to  permit 
the  flagella  to  show  well,  so  that  it  is  recommended  to  prepare  a  second  by  plac- 
ing a  small  drop  of  distilled  water,  upon  a  cover  and  taking  a  loopful  from  the 
first  dilution  to  make  the  second,  and  spreading  it  over  the  entire  surface  without 
much  rubbing  or  stirring.  The  film  is  allowed  to  dry,  and  is  then  fixed  by  passing 
it  three  times  through  the  flame.  When  this  is  done  with  forceps  there  is  some 
danger  of  the  preparation  becoming  too  hot,  so  Lofifler  recommends  that  the  glass 
be  held  in  the  fingers  while  the  passes  through  the  flame  are  made. 

The  cover-glass  is  now  held  in  forceps,  and  the  mordant,  solution  A,  dropped 
upon  it  until  it  is  well  covered,  when  it  is  warmed  until  it  begins  to  steam.  The 
mordant  must  be  replaced  as  it  evaporates.  It  must  not  be  heated  too  strongly: 
above  all  things,  must  not  boil.  This  solution  is  allowed  to  act  from  one-half 
to  one  minute,  is  then  washed  off  with  distilled  water,  and  then  with  absolute 
alcohol  until  all  traces  of  the  solution  have  been  removed.  The  real  stain — 
Lofifler  recommends  an  anilin-water  fuchsin  (Ehrlich's  solution) — which  should 
have  a  neutral  reaction,  is  next  dropped  on  so  as  to  cover  the  film,  and  heated  for 
a  minute  until  vapor  begins  to  rise,  after  which  it  is  washed  off  carefully,  dried, 
and  mounted  in  Canada  balsam.  To  obtain  the  neutral  reaction  of  the  stain, 
enough  of  the  i  per  cent,  sodium  hydrate  solution  is  added  to  an  amount  of  the 
anilin-water-fuchsin  solution  having  a  thickness  of  several  centimeters  to  begin 
to  change  the  transparent  into  an  opaque  solution. 

A  specimen  thus  treated  may  or  may  not  show  the  flagella.  If  not,  before 
proceeding  further  it  is  necessary  to  study  the  chemic  products  of  the  micro- 
organism in  culture  media.  If  by  its  growth  the  organism  elaborates  alkalies, 
from  i  drop  to  i  cc.  of  solution  C  in  16  cc.  must  be  added  to  the  mordant  A,  and 
the  staining  repeated.  It  may  be  necessary  to  stain  again  and  again  until  the 
proper  amount  is  determined  by  the  successful  demonstration  of  the  flagella. 
On  the  other  hand,  if  the  organism  by  its  growth  produces  acid,  solution  B  must 
be  added,  drop  by  drop,  and  numerous  stained  specimens  examined  to  see  with 
what  addition  of  alkali  the  flagella  will  appear.  Loffler  fortunately  worked  out 
the  amounts  required  for  some  species,  and  of  the  more  important  ones  the  fol- 
lowing solutions  of  B  and  C  must  be  added  to  16  cc.  of  solution  A  to  attain  the 
desired  effect: 

Cholera  spirillum J^-i  drop  of  solution  C 

Typhoid  fever i  cc.  of  solution  B 

Bacillus  subtilis 28-30  drops  of  solution  B 

Bacillus  of  malignant  edema .  36  or  37  drops  of  solution  B 

Part  of  the  success  of  the  staining  depends  upon  using  a  very  young 
culture  and  having  the  bacteria  thinly  spread  upon  the  glass,  so 
as  to  be  as  free  from  albuminous  and  gelatinous  materials  as  possible. 
The  cover-glass  must  be  cleaned  most  painstakingly;  too  much 
heating  in  fixing  must  be  avoided.  After  using  and  washing  off 
the  mordant,  the  preparation  should  be  dried  before  the  applica- 
tion of  the  anilin-water-fuchsin  solution. 

*  Ibid.,  1890,  Bd.  vii,  p.  625. 


Staining  159 

PitfielcTs  Method. — Pitfield*  has  devised  a  single  solution,  at  once 
mordant  and  stain.  It  is  made  in  two  parts,  which  are  filtered  and 
mixed : 

(A)- 

Saturated  aqueous  solution  of  alum 10  cc. 

Saturated  alcoholic  solution  of  gentian  violet i  " 

(B)- 

Tannic  acid i  gram 

Distilled  water 10  cc. 

The  solutions  should  be  made  with  cold  water,  and  immediately 
after  mixing  the  stain  is  ready  for  use.  The  cover-slip  is  carefully 
cleaned,  the  grease  being  burned  off  in  a  flame.  After  it  has  cooled, 
the  bacteria  are  spread  upon  it,  well  diluted  with  water.  After 
drying  thoroughly  in  the  air,  the  stain  is  gradually  poured  on  and 
by  gentle  heating  brought  almost  to  a  boil;  the  slip  covered  with 
the  hot  stain  is  laid  aside  for  a  minute,  then  washed  in  water  and 
mounted. 

Smith's  Modification  of  Pitfield' s  Method.^ — A  boiling  saturated  solution  of 
bichlorid  of  mercury  is  poured  into  a  bottle  in  which  crystals  of  alum  have 
been  placed  in  quantity  more  than  sufficient  to  saturate  the  fluid.  The 
bottle  is  shaken  and  allowed  to  cool;  10  cc.  of  this  solution  are  added  to 
the  same  volume  of  freshly  prepared  tannic  acid  solution  and  5  cc.  of  car- 
bol  f  uchsin  added.  Mix  and  filter.  The  filtrate,  which  is  the  mordant,  is 
caught  directly  upon  the  spread  (the  liquid  must  always  be  filtered  at  the 
time  of  use)  and  heated  gently  for  three  minutes,  but  not  permitted  to 
boil.  Wash  with  water  and  then  stain  in  the  following: 

Saturated  alcoholic  solution  of  gentian  violet i  cc. 

Saturated  solution  of  ammonium  alum 10    ' 

Filter  the  stain  directly  upon  the  slide  at  the  time  of  using,  and  heat  it 
for  three  to  four  minutes.  Wash  thoroughly  in  water,  dry,  and  mount 
in  balsam. 

Van  Ermengem's  Method. — Van  Ermengemt  has  devised  a  some- 
what complicated  method  of  staining  flagella,  which  has  given  great 
satisfaction.  Three  solutions,  which  he  describes  as  the  bain 
fixateur,  bain  sensibilisateur,  and  bain  reducteur  et  reinforqateur,  are 
to  be  used  as  follows: 

i.  Bain  fixateur: 

2  per  cent,  solution  of  osmic  acid i  part 

10-25  Per  cent-  solution  of  tannin 2  parts 

The  cover-glasses,  which  are  very  thinly  spread,  dried,  and  fixed, 
are  placed  in  this  bath  for  one  hour  at  the  room  temperature,  warmed 
until  steam  arises,  and  then  kept  hot  for  five  minutes.  They  are 
next  washed  with  distilled  water,  then  with  absolute  alcohol,  then 
again  with  distilled  water.  All  three  washings  must  be  very 
thorough. 

*  "Medical  News,"  Sept.  7,  1895. 
t  "British  Medical  Journal,"  1901,  I,  p.  205. 

J  "Travaux  du  Lab.  d'hygiene  et  des  bact.  de  Gand.,"  t.  I,  p.  3.  Abstracted 
in  the  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1894,  Bd.  xv,  p.  969. 


160  Methods  of  Observing  Micro-organisms 

2.  Bain  sensibilisateur: 

5  per  cent,  solution  of  nitrate  of  silver  in  distilled  water. 

The  films  are  allowed  to  remain  in  this  for  a  few  seconds,  and  are 
then  immediately  transferred  to  the  third  bath. 
3.  Bain  reducteur  et  reinforqateur: 

Gallic  acid 5  grams 

Tannin 3       " 

Fused  potassium  acetate 10      " 

Distilled  water 350  cc. 

The  preparations  are  kept  in  this  solution  for  a  few  seconds,  then 
returned  to  the  nitrate  of  silver  solution  until  they  begin  to  turn 
black.  They  are  then  washed,  dried,  and  mounted. 

Mervyn  Gorden  modifies  the  method  by  allowing  the  preparations 
to  remain  in  the  second  bath  for  two  minutes,  transferring  to  the 
third  bath  for  one  and  a  half  to  two  minutes,  and  then  washing, 
drying,  and  mounting  without  returning  to  the  second  bath. 

Muir  and  Ritchie  find  it  advantageous  to  use  a  fresh  supply  of 
the  third  solution  for  each  specimen. 

Rossi*  gives  the  following  directions  for  staining  flagella: 

The  culture  to  be  examined  should  be  a  young  culture,  not  more  than  ten, 
eighteen,,  or  twenty-four  hours  old.  It  should  be  made  upon  freshly  prepared 
agar-agar,  or  upon  the  reagent  after  it  has  been  melted  and  then  congealed, 
as  it  is  of  the  utmost  importance  that  the  surface  be  moist.  The  culture 
should  be  examined  by  the  hanging-drop  method  to  see  that  the  organisms 
are  actively  motile  before  the  staining  is  attempted. 

The  staining  should  be  done  only  after  the  greatest  care  has  been  taken  to 
see  that  all  the  conditions  are  favorable.  For  this  reason  the  cover-glasses  em- 
ployed in  making  the  spreads  must  be  carefully  cleaned  with  alcohol,  then 
immersed  in  steaming  sulphuric  acid  for  ten  to  fifteen  minutes.  They  are  then 
washed  in  water,  then  placed  in  a  mixture  of  alcohol  and  benzine  (equal  parts), 
wiped  with  a  clean  soft  cloth,  and  passed  through  the  colorless  Bunsen  flame 
forty  to  fifty  times,  and  then  that  side  of  the  glass  utilized  for  the  " spread"  that 
has  been  in  direct  contact  with  the  flame. 

A  platinum  loopful  of  the  appropriate  culture  is  placed  in  a  drop  of  distilled 
water  upon  a  clean  slide  and  slightly  stirred.  If  conditions  are  favorable,  it  forms 
a  homogeneous  emulsion.  If  clumps  appear,  the  cultural  conditions  are  not 
favorable. 

If  favorable,  a  loopful  of  this  dilution  is  added  to  i  cc.  of  distilled  water  in  a 
clean  cover-glass  and  thoroughly  stirred.  From  the  center  of  the  surface  of  this 
fluid  a  platinum  loopful  is  next  taken  and  placed  upon  each  of  the  prepared 
cover-glasses  and,  without  spreading  or  stirring,  allowed  to  dry  in  the  air  or  in  an 
exsiccator. 

The  staining  solutions  are  made  as  follows: 

(A)  A  solution  of  50  grams  of  pure  crystalline  carbolic  acid  in  1000  cc.  of 

distilled  water,  to  which  40  grams  of  pure  tannin  are  added,  the  whole 
being  warmed  on  a  water-bath  until  solution  is  complete. 

(B)  Basic  fuchsin  (rosanilinchlorhydrate) 2.5  grams 

Absolute  alcohol 100 .  o  cc. 

(C)  Potassium  hydrate i  •  o  gram 

Distilled  water 100 .  o  grams 

Mix  solutions  A  arid  B  and  preserve  in  a  well-closed  bottle.  Place  solution  C 
in  a  bottle  with  a  pipette  stopper.  When  the  staining  is  to  be  done,  one  pours 
15  to  20  ce.  of  the  "A  B  mixture  into  a  glass-stoppered  test-tube  and  adds  2  or  3 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Orig.,  1903,  xxxm,  p.  572 


The  Observation  of  Living  Protozoa  161 

drops  of  solution  C.  A  precipitate  forms,  but  quickly  dissolves  on  shaking. 
More  of  solution  C  is  added,  and  the  tube  shaken  until  the  solution  becomes 
brown  and  clouded  and  one  can  see  a  fine  precipitate  in  a  thin  layer  of  the  fluid. 
The  fluid  is  next  filtered  several  times  through  the  same  filter  and  caught  in  the 
same  glass  until  it  will  remain  clear  for  several  minutes.  Then  it  is  poured  on  the 
filter  a  last  time  and  4  or  5  drops  allowed  to  fall  upon  each  of  the  prepared  cover- 
glasses.  In  a  short  time  a  sheen  is  observed  upon  the  surface  of  the  fluid  on  the 
cover-glasses,  showing  that  a  fine  precipitate  has  formed.  When  this  has 
occurred,  a  little  experience  will  show  when  the  proper  moment  arrives  to  throw 
off  the  fluid  and  wash  the  cover  in  distilled  water.  It  is  the  precipitate  that  clings 
to  the  flagella  and  renders  them  distinctly  visible.  If  no  precipitate  occurs,  the 
flagella  will  not  be  seen. 

L.  Smith*  offers  the  following  modification  of  Newman's  method f 
as  being  a  simple  and  excellent  method  of  staining  flagella: 

The  material  and  cover-glasses  are  prepared  with  care  as  for  the 
foregoing  methods,  after  which  one  proceeds  as  follows: 

1.  Transfer  a  loopful  of  the  bacillary  emulsion  to  the  clean  slide  or  cover- 

glass  and  allow  it  to  dry  in  the  air. 

2.  Expose  to  a  mild  degree  of  heat,  holding  the  glass  in  the  fingers — this  is 

rather  drying  than  actual  heating. 

3.  Allow  the  stain  to  drop  from  a  filter  upon  the  film  and  remain  in  contact 

five  to  ten  minutes. 
The  formula  for  the  stain  is 

I.  Tannic  acid '. i  gram 

Potassium  alum •.      i     " 

Distilled  water 40  cc. 

Dissolve  by  shaking  or  allow  to  stand  overnight  in  the  incubator. 

II.  "Night blue"} 0.5  gram 

95  per  cent,  or  absolute  alcohol 20.0  cc. 

Mix  I  and  II  thoroughly  and  remove  the  heavy  precipitate  by  filtration. 
If  not  used  at  once,  drop  from  a  filter  upon  the  film.  The  stain  does 
not  keep  more  than  a  few  days. 

4.  Wash  carefully  but  thoroughly  in  water. 

5.  Apply  a  saturated  aqueous  solution  of  gentian  violet  for  about  two 

minutes  to  stain  the  bodies  of  the  bacteria. 

6.  Wash  thoroughly  in  water,  dry  with  smooth  blotting-paper,  and  mount 

in  balsam. 

To  secure  a  perfectly  clean  background  for  photomicrography,  it  is  best  to 
stain  on  a  slide.  The  stain  is  then  poured  into  a  Petri  dish,  the  slide  inverted,  the 
end  of  the  slide  used  to  push  aside  the  film  on.  the  surface  of  the  stain,  and  the 
film  then  immersed  downward,  one  end  of  th'e  slide  supported,  during  staining, 
on  a  match-stick  or  bit  of  glass  rod.  In  this  way  the  adherence  of  the  precipitate 
to  the  slide  can  be  avoided. 


THE  OBSERVATION  OF  LIVING  PROTOZOA 

When  protozoa  are  to  be  examined  in  transparent  fluids,  such  as 
pond-water  or  culture  fluids  in  which  they  have  been  artificially 
nourished,  use  can  be  made  of  a  "live-box"  or  of  the  "hanging  drop." 
Ordinarily,  however,  the  organisms  to  be  examined  are  contained 
in  blood,  in  pus,  in  sputum,  in  feces,  or  in  some  other  more  or  less 
opaque  fluid,  of  which  an  extremely  thin  layer  must  be  prepared  in 
order  that  the-  formed  elements  may  be  separated  sufficiently  for 
the  individual  cells  and  organisms  to  be  seen. 

*  "Jour.  Med.  Research,"  1901,  vi,  p.  341.  ^ 

t  "Bacteria,"  John  Murray,  London,  2d  edition. 

}  James  Strong  &  Son,  Glasgow  and  Manchester. 


1 62  Methods  of  Observing  Micro-organisms 

Such  a  thin  layer  is  usually  easily  obtained  by  the  use  of  a  slide 
and  cover-glass,  and  the  careful  preparation  of  a  good  film. 

The  slide  and  the  cover-glass  should  be  thoroughly  cleansed  and 
freed  from  fat  and  grit  and  well  polished.  A  comparatively  small 
drop  of  blood — let  us  say,  for  example — is  placed  upon  the  center 
of  the  slide  and  immediately  covered  with  the  cover-glass.  If  the 
drop  is  not  too  large  and  the  glasses  are  clean,  the  weight  of  the  cover- 
glass  causes  the  drop  to  spread,  and  capillary  attraction  completes 
the  formation  of  a  very  thin  film.  The  quantity  of  blood  used 
should  not  be  sufficient  to  reach  the  edges  of  the  cover-glass,  else 
sometimes  the  glass  is  pressed  up  instead  of  being  drawn  down  and 
moves  about  too  freely.  If  the  examination  is  to  take  enough  time 
to  cause  the  drop  to  dry,  a  match-stick  dipped  in  thin  vaselin  and 
drawn  about  the  edge  of  the  cover  will  prevent  it. 

Such  a  film  is  usually  best  examined  at  or  near  the  center,  where 
the  formed  elements  are  not  widely  separated. 

The  living  protozoa  in  preparations  of  this  kind  may  be  examined 
by  ordinary  illumination  by  transmitted  light,  or  with  lateral 
illumination  by  means  of  the  "dark-field  illuminator."  The  latter 
serves  better  for  the  discovery  of  the  very  small  transparent  organ- 
isms — spirochaeta  and  treponema — and  for  the  observation  of  the 
cilia  and  flagella. 

STAINING  PROTOZOA 

It  is  through  the  study  of  stained  protozoa  that  we  arrive  at  most 
of  our  knowledge  of  their  structural  details.  They  can  be  stained 
in  blood  or  fluids  upon  a  slide  or  in  sections  of  tissue. 

As  in  the  case  of  the  bacteria,  it  is  first  necessary  to  prepare 
satisfactory  spreads  for  the  purpose.  In  order  that  the  description 
shall  be  as  practical  as  possible,  we  will  suppose  that  the  micro- 
organisms to  be  stained  are  in  blood — spirochaeta,  plasmodium,  etc. 

As  pointed  out  above,  the  protozoa,  under  such  circumstances, 
are  distributed  among  or  in  cellular  elements  that  interfere  with 
satisfactory  observation  unless  precautions  are  taken  to  separate 
them  as  widely  as  may  be  required. 

1.  Cover- glasses. — The  glasses  should  be  perfectly  clean  and  freed  from  fat, 

either  by  washing  in  alcohol  and  ether  and  wiping  with  a  clean  soft 
cotton  cloth  or  Chinese  rice  paper,  or  by  flaming.  The  drop  of  blood 
should  be  small  and  should  be  placed  upon  the  center  of  one  glass  and 
immediately  covered  by  another,  so  held  that  the  corners  do  not 
coincide.  As  soon  as  the  drop  is  fairly  well  distributed  the  glasses  are 
gently  slid  apart. 

2.  Slides. — The  slides,  like  the  cover-glasses,  must  be  perfectly  clean.     The 

drop  of  blood  is  placed  upon  one  slide  at  about  one-fourth  the 
length  of  the  slide  from  its  end,  touched  with  the  end  (it  must  have 
ground  edges)  of  the  second  slide,  and  then  gently  pushed  along  until 
the  fluid  is  exhausted. 

If  the  covers  are  to  be  stained,  they  can  most  conveniently  be 
held  in  the  Stewart  forceps.  If  the  slides  are  used,  they  can  be 
held  in  the  fingers. 


.     Staining  Protozoa  163 

The  stain  most  useful  is  that  of  Romanowsky.  It  has  many 
modifications,  of  which  the  most  used  and  best  known  are  Giemsa's, 
Jenner's,  Leishman's,  Wright's,  and  Marino's.  These  stains  can 
be  bought  either  in  solution  or  in  tablet  form  ready  for  solution. 

Those  most  highly  to  be  recommended  are  Wright's  and  Marino's. 


Fig-  35. — Method  of  making  dry  film  with  two  cover-glasses  (from  Daniels' 
"Laboratory  Studies  in  Tropical  Medicine"). 

Wright's  Blood-stain. — This  is  a  modification  of  Leishmann's  stain,  to  which 
it  is  to  be  preferred  because  it  can  be  made  in  a  few  hours  instead  of  eleven 
days.  It  combines  the  methylene-blue-eosin  combination  of  Roman- 
owsky with  the  methyl-alcohol  fixation  of  Jenner. 

It  is  prepared  as  follows:* 

"To  a  0.5  per  cent,  aqueous  solution  of  sodium  bicarbonate  add  methylene- 
blue  (B.  X.  or  "medicinally  pure")  in  the  proportion  of  i  gm.  of  the  dye 


Fig.  36. — Method  of  making  dry  films  with  two  slides  (from  Daniels' 
tory  Studies  in  Tropical  Medicine"). 


'Labora- 


to  100  cc.  of  the  solution.  Heat  the  mixture  in  a  steam  sterilizer  at 
ioo°C.  for  one  full  hour,  counting  the  time  after  the  sterilizer  has  become 
thoroughly  heated.  The  mixture  is  to  be  contained  in  a  flask  of  such  size 
and  shape  that  it  forms  a  layer  not  more  than  6  cm.  deep.  After  heating, 
the  mixture  is  allowed  to  cool,  placing  the  flask  in  cold  water  if  desired,  and 

*  Mallory  and  Wright,  "Pathological  Technique,"  1911,  p.  364. 


164  Methods  of  Observing  Micro-organisms 

is  then  filtered,  to  remove  the  precipitate  which  has  formed  in  it.  It 
should,  when  cold,  have  a  deep  purple-red  color  when  viewed,  in  a  thin 
layer,  by  transmitted  yellowish  artificial  light.  It  does  not  show  this 
color  while  it  is  warm.  To  each  100  cc.  of  the  filtered  mixture  add  500 
cc.  of  a  o.i  per  cent,  aqueous  solution  of  "yellowish,  water-soluble"  eosin 
and  mix  thoroughly.  Collect  the  abundant  precipitate  which  immediately 
appears  on  a  filter.  When  the  precipitate  is  dry,  dissolve  it  in  methylic 
alcohol  (Merck's  "reagent")  in  the  proportion  of  o.i  gr.  to  60  cc.  of  the 
alcohol.  In  order  to  facilitate  the  solution  the  precipitate  is  to  be  rubbed 
up  with  the  alcohol  in  a  porcelain  dish  or  mortar  with  a  spatula  or  pestle. 
"This  alcoholic  solution  of  the  precipitate  is- the  staining  fluid.  It  should 
be  kept  in  a  well-stoppered  bottle  because  of  the  volatility  of  the  alcohol. 
If  it  becomes  too  concentrated  by  evaporation,  and  thus  stains  too 
deeply  or  forms  a  precipitate  on  the  blood-smear,  the  addition  of  a  suitable 
quantity  of  methylic  alcohol  will  quickly  correct  such  fault.  It  does  not 
undergo  any  other  spontaneous  change  than  that  of  concentration  by 
evaporation." 

Method  of  Staining.— The  blood-films  are  permitted  to  dry  in  the  air  (not 
heated) : 

1.  Cover  the  film  with  a  noted  quantity  of  the  staining  fluid  by  means  of  a 

medicine  dropper. 

2.  After  one  minute  add  to  the  staining  fluid  the  same  quantity  of  distilled 

water  by  means  of  the  medicine  dropper,  and  allow  it  to  remain  for  two 
or  three  minutes,  according  to  the  intensity  of  the  staining  desired. 
A  longer  period  of  staining  may  produce  a  precipitate. 

3.  Wash  the  preparation  in  water  for  thirty  seconds  or  until  the  thinner 

portions  of  the  preparation  become  yellow  or  pink  in  color. 

4.  Dry  and  mount  in  balsam. 

Films  more  than  an  hour  old  do  not  stain  so  well  as  fresh  ones.  Old  films 
show  bluish  instead  of  pink  erythrocytes. 

Marino's  stain*  is  extremely  delicate  and  gives  still  more  beautiful  results 
where  parasites  are  present.  It  is  an  azur-eosin  combination,  prepared  as 
follows : 

Solution  I: 

Methylene-blue  (medicinal) 0.5  gram 

Azurll 0.5     " 

Water  (distilled) 100 .  o  cc. 

Solution  II: 

Sodium  carbonate 0.5  gram 

Water 100.0  cc. 

Pour  the  two  solutions  together  and  stand  the  mixture  in  the  thermostat 
for  forty-eight  hours  at  37°C.;  then  add  0.2  per  cent,  aqueous  solution  of 
eosin  ("yellowish  aqueous  eosin").  The  quantity  of  this  solution  must 
be  varied  according  to  the  blue  dyes  employed,  so  as  to  secure  the  maxi- 
mum precipitation.  The  exact  quantity  can  only  be  determined  by 
titration.  A  precipitate  now  forms  in  the  course  of  twenty-four  hours. 
This  is  caught  upon  a  filter-paper  and  dried. 

The  precipitate,  dissolved  in  methylic  alcohol,  in  the  proportion  of  0.04  gm. 
of  the  powder  to  20  cc.  of  the  methylic  alcohol,  forms  the  stain. 

Method. — The  stain  is  dropped  upon  the  spread  so  as  to  cover  it,  the  number 
of  drops  being  counted.  It  is  permitted  to  act  for  exactly  three  minutes 
for  purposes  of  fixation,  then,  without  pouring  off  the  stain,  twice  the 
number  of  drops  of  a  i  :  100,000  aqueous  eosin  solution  are  added.  |  The 
two  fluids  gradually  mix,  transfusion  currents  are  formed,  and  the  speci- 
men is  allowed  to  stand  for  exactly  two  minutes  longer.  It  is  during  this 

*  "Ann.  de  PInst.  Pasteur,"  1904,  xvm,  761. 

f  Marino  used  a  i  :  20,000  aqueous  solution  of  eosin,  but  the  i  :  100,000 
solution  is  less  apt  to  cause  objectionable  precipitation  of  the  dye  and  gives 
equally  good  results. 


Staining  Protozoa  in  Tissue  ¥65 

time  that  the  staining  takes  place.  A  precipitate  usually  forms  upon  the 
surface  of  the  fluid,  so  that  it  must  not  be  poured  off,  but  splashed  off 
by  dropping  distilled  water  upon  it  from  a  height.  The  distilled  water 
is  added  until  it  no  longer  shows  any  color,  when  the  specimen  is  drained, 
dried,  and  mounted  in  balsam. 

The  student  may  also  try  staining  with  hematoxylin  and  eosin, 
thionin  and  eosin,  methylene-blue  and  eosin,  or  any  other  dyes, 
some  of  which  sometimes  bring  out  special  details  of  structure. 
The  protozoa  do  not  show  the  same  reaction  to  Gram's  stain  that 
makes  it  so  useful  for  differentiating  the  bacteria. 


STAINING  PROTOZOA  IN  TISSUE 

For  this  purpose  the  sections  should  be  embedded  in  paraffin, 
cut  very  thin,  and  cemented  to  the  slides. 

Ordinary  staining  with  hematoxylin  and  eosin  is  rarely  of  much 
use.  Methylene-blue  and  eosin  is  better,  but  still  more  useful  are 
the  Romanowsky  methods,  and  both  the  Wright  stain  and  the 
Marino  stain  can,  with  some  modification  of  the  time  of  staining 
and  washing,  be  employed  with  good  results. 

Still  better  and  more  satisfactory  for  certain  protozoa  are  the 
iron-hematoxylin  and  the  Biondi  stain. 

Heidenhain's  Iron-hematoxylin.* — Fix  the  tissue,  by  preference,  in  Zenker's 
solution,  though  alcohol  fixation  will  do.  Embed  in  paraffin,  cut  very 
thin,  and  fix  to  the  slide. 

1.  Stain  from  three  to  twelve  hours  in  2.5  per  cent,  solution  of  violet  iron- 

alum  (sulphate  of  iron  and  ammonium).  The  sections  should  be 
stood  vertically  in  the  solution,  so  that  no  precipitate  may  form 
upon  them. 

2.  Wash  quickly  in  water. 

3.  Stain  in  a  0.5  per  cent,  ripened  alcoholic  solution  of  hematoxylin  for 

from  twelve  to  thirty-six  hours. 

4.  Wash  in  water. 

5.  Differentiate  in  the  iron-alum  solution,  controlling  the  results  under  the 

microscope.  The  section  should  be  well  washed  in  a  large  dish  of  tap- 
water  before  each  examination  to  stop  decolorization. 

6.  Wash  in  running  water  for  a  quarter  of  an  hour. 

7.  Pass  through  alcohol,  xylol,  and  mount  in  xylol  balsam. 

A  counterstain  with  Bordeau  R.  before  or  with  rubin  S.  after  the  iron  stain 
is  sometimes  useful. 

Biondi-H eidenhain  Stain,  f — The  tissues  must  be  fixed  in  Zenker's  or  corrosive 
sublimate  solutions.  Embed  in  paraffin,  cut  very  thin,  fix  to  the  slide. 

Stain     I.  Orange  G 8  grams 

Water 100  cc. 

II.  Acid  fuchsin    1  20  s 

or  Rubin  S.      / 
Water 100  cc. 

III.  Methyl-green 8  grams 

Water 100  cc. 

Let  the  solutions  stand  for  several  days,  occasionally  shaking  the  bottles 
to  make  sure  that  a  saturated  solution  of  each  is  secured.  At  the 
end  of  the  time  set,  mix  the  solutions  in  the  following  proportions: 

*  Mallory  arid  Wright,  "Pathological  Technique,"  1911,  p.  309. 

f  Modified  from  Mallory  and  Wright,  "Pathological  Technique,"  1911,  p.  289. 


1 66  Methods  of  Observing  Micro-organisms 

I ioo  parts 

II 20     " 

III 50     " 

At  the  time  of  staining  dilute  the  mixture  i  :6o  or  i  :  ioo  with  water. 
To  test  the  solution:  (i)  Acetic  acid  makes  it  redder.  (2)  A  drop  of 
the  solution  on  filter-paper  should  make  a  blue  spot  with  a  green 
center  and  an  orange  border.  If  a  red  zone  appears  outside  of  the 
orange,  too  much  acid  fuchsin  is  present. 

1.  Stain  the  sections  from  six  to  twenty-four  hours. 

2.  Wash  out  a  little  in  90  per  cent,  alcohol. 

3.  Dehydrate  in  absolute  alcohol. 

4.  Xylot. 

5.  Xylol  balsam. 

It  is  important  to  place  the  sections  directly  from  the  staining  fluid  into  the 
alcohol,  because  water  instantly  washes  out  the  methyl-green. 

Ross'  Thick  Blood-spreads. — In  case  the  number  of  parasites  in 
the  blood  is  very  small,  so  that  they  would  be  scattered  sparingly 
over  a  large  area  of  the  ordinary  blood  spread,  Ross*  has  suggested 
a  modification  of  the  technic  by  which  they  can  be  more  readily  found. 
To  do  this  a  very  thick  spread  is  prepared  and  dried.  As  soon  as 
it  is  dry,  and  without  fixing,  the  slide  is  stood  vertically  in  a  vessel 
filled  with  distilled  water.  The  red  corpuscles  at  once  begin  to 
hemolyze  and  the  process  is  carried  on  to  completion.  When  all 
of  the  hemoglobin  has  been  removed,  the  slide  is  taken  out,  dried, 
and  then  fixed  and  stained.  There  now  being  no  red  corpuscles  to 
distract  the  attention  or  obscure  the  vision,  the  stained  parasites 
can  quickly  be  found. 

Measurement  of  Micro-organisms. — They  can  best  be  measured 
by  an  eyepiece  micrometer.  As  these  instruments  vary  somewhat 
in  construction,  the  unit  of  measurement  for  each  objective  magni- 
fication and  the  method  of  manipulating  the  instruments  must  be 
learned  from  dealers'  catalogues. 

Photographing  Micro-organisms. — This  requires  special  apparatus 
and  methods,  for  which  it  is  necessary  to  refer  to  special  text-books,  f 

*  "Lancet,"  Jan.  10,  1903. 

f  See  the  excellent  chapter  upon  Photomicrography  in  Aschoff  and  Gaylord's 
"Pathological  Histology,"  Philadelphia,  1900. 


CHAPTER  VI 
STERILIZATION  AND  DISINFECTION 

BEFORE  considering  the  methods  employed  for  the  artificial 
cultivation  of  micro-organisms  and  for  the  preparation  of  media 
for  that  purpose,  it  is  necessary  to  have  a  thorough  knowledge  of 
the  principles  of  sterilization  and  disinfection  in  order  intelligently 
to  apply  the  methods  to  the  elimination  or  destruction  of  micro- 
organisms whose  accidental  presence  might  ruin  the  experiments. 

The  dust  of  the  atmosphere,  almost  invariable  in  its  micro- 
organismal  contamination,  constantly  settles  upon  our  glassware, 
pots,  kettles,  funnels,  etc.,  and  would  certainly  ruin  every  culture- 
medium  with  which  we  experiment  did  we  not  take  appropriate 
measures  for  its  purification  and  protection. 

To  get  rid  of  these  undesirable  "weeds"  we  make  use  of  our 
knowledge  of  the  conditions  destructive  to  bacterial  life,  and  sub- 
ject the  articles  contaminated  by  them  to  the  action  of  heat  beyond 
their  known  enduring  power,  or  to  the  action  of  chemic  agents  known 
to  destroy  them,  or  remove  them  from  fluids  into  which  they  have 
entered  by  passage  through  unglazed  porcelain.  By  all  of  these 
methods  the  articles  are  made  sterile.  Anything  is  sterile  when  it 
contains  no  germs  of  life. 

Sterilization  is  the  act  of  making  sterile  by  destroying  or  re- 
moving all  micro-organismal  life,  whether  infectious  or  non-in- 
fectious. Disinfection  signifies  the  destruction  of  the  infectious 
agents,  taking  no  account  of  those  that  are  non-infectious.  A 
germicide  is  any  substance  that  will  kill  germs.  It  may  be  used  for 
disinfection  and  for  sterilization.  An  antiseptic  is  a  substance  that 
will  inhibit  the  growth  of  micro-organisms.  It  does  not  necessarily 
kill  them. 

The  following  table  will  serve  to  outline  the  methods  used  for 
effecting  sterilization  or  the  complete  destruction  or  removal  of 
living  organisms: 

I.  The  Sterilization  and  Protection  of  Instruments  and  Glassware. 
— Sterilization  may  be  accomplished  by  either  moist  or  dry  heat. 
For  the  perfect  sterilization  of  objects  capable  of  withstanding  it, 
tubes,  flasks,  dishes,  etc.,  dry  heat  is  always  to  be  preferred,  because 
of  its  more  certain  action.  If  we  knew  just  what  organisms  we  had 
to  deal  with,  we  might  be  able  in  many  cases  to  save  time  and  gas; 
but  though  some  non-spore-producing  forms  are  killed  at  a  tem- 
perature of  6o°C.,  spore-bearers  may  withstand  ioo°C.  .for  an  hour; 

167 


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Sterilization  and  Disinfection 


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Methods  of  Sterilization 


169 


it  is,  therefore,  best  to  employ  a  temperature  high  enough  to  kill 
all  with  certainty.  The  apparatus  is  known  as  a  "  hot-air  sterilizer." 
Platinum  wires  used  for  inoculation  are  sterilized  by  being  held 
in  the  direct  flame  until  they  become  incandescent.  In  sterilizing 
the  wires  attention  must  be  bestowed  upon  the  glass  handle,  which 
should  be  flamed  for  least  half  its  length  for  a  few  moments. 
Carelessness  in  this  respect  may  result  in  the  contamination  of  the 
cultures. 


Fig.  37.— Hot-air  sterilizer.  The  gas  jets  are  inclosed  within  the  space 
between  the  outer  and  middle  walls,  C,  and  can  be  seen  at  F.  That  heat  ascends, 
warming  the  air  between  the  two  inner  walls,  which  ascends  between  the  walk, 
Ky  then  descends  over  the  contents,  /,  and  escapes  through  perforations  in  the 
bottom,  B,  to  supply  the  draft  at  F,  and  eventually  escapes  again  at  S;  R,  gas 
regulator;  T,  thermometer. 

Knives,  scissors,  and  forceps  may  be  exposed  for  a  very  brief 
time  to  the  direct  flame,  but  as  this  affects  the  temper  of  the  steel 
when  continued  too  long,  they  are  better  boiled,  steamed  or 
carbolized. 

All  articles  of  glassware  are  to  be  sterilized  by  an  exposure  of 
one-half  to  one  hour  to  a  sufficiently  high  temperature—  i5o°C.  or 
302°F. — in  the  hot-air  sterilizer.  This  temperature  is  fatal  to  all 
forms  of  microscopic  life. 

Rubber  stoppers,  corks,  wooden  apparatus,  and  other  objects  which 
are  warped,  cracked,  charred,  or  melted  by  so  high  a  temperature 


I  jo  Sterilization  and  Disinfection 

must  be  sterilized  by  exposure  to  streaming  steam  or  steam  under 
pressure,  in  the  steam  sterilizer  or  autoclave,  before  they  can  be 
pronounced  sterile. 

It  must  always  be  borne  in  mind  that  after  sterilization  has  been 
accomplished  it  is  necessary  to  protect  the  sterilized  objects  and 
media  from  future  contamination. 

To  Schroder  and  Van  Dusch  belongs  the  credit  of  having  first 
shown  that  when  the  mouths  of  flasks  and  tubes  are  closed  with 
plugs  of  sterile  cotton  no  germs  can  filter  through.  This  discovery 
has  been  of  inestimable  value,  and  has  been  one  of  the  chief  means 
permitting  the  advance  of  bacteriology.  If,  before  sterilizing, 
flasks  and  tubes  are  carefully  plugged  with  ordinary  (non-absorbent) 
cotton- wool,  they  and  their  contents  will  remain  free  from  the 
access  of  germs  until  opened.  Instruments  may  be  sterilized  wrapped 
in  cotton,  to  be  opened  only  when  ready  for  use ;  or  instruments 
and  rubber  goods  sterilized  by  steam  can  subsequently  be  wrapped 
in  sterile  cotton  and  kept  for  use.  It  is  of  the  utmost  importance  to 
carefully  protect  every  sterilized  object,  in  order  that  the  object  of 
the  sterilization  be  not  defeated.  As  the  spores  of  molds  falling 
upon  cotton  sometimes  grow  and  allow  their  mycelia  to  work  their 
way  through  and  drop  into  the  culture-medium,  Roux  has  em- 
ployed paper  caps,  with  which  the  cotton  stoppers  can  be  pro- 
tected from  the  dust.  These  are  easily  made  by  curling  a  small 
square  of  paper  into  a  "cornucopia,"  and  fastening  by  turning  up 
the  edge  or  putting  in  a  pin.  The  paper  is  placed  over  the  stopper 
before  the  sterilization,  after  which  no  contamination  of  the  cotton 
can  occur. 

II.  Sterilization  and  Protection  of  Culture-media. — As  almost 
all  of  the  culture-media  contain  about  80  per  cent,  of  water,  which 
would  evaporate  in  the  hot-air  closet,  and  so  destroy  the  material, 
hot-air  sterilization  is  inappropriate  for  them,  sterilization  by  stream- 
ing steam  being  the  only  satisfactory  method.  The  prepared 
media  are  placed  in  previously  sterilized  flasks  or  tubes,  carefully 
plugged  with  cotton-wool,  and  then  sterilized  in  an  Arnold's  steam 
sterilizer. 

The  temperature  of  boiling  water,  ioo°C.,  does  not  kill  the  spores, 
so  that  one  exposure  of  the  culture-media  to  streaming  steam  is  of 
little  use.  The  sterilization  must  be  applied  in  a  systematic 
manner — intermittent  sterilization — based  upon  a  knowledge  of 
sporulation. 

In  carrying  out  intermittent  sterilization  the  culture-medium 
is  exposed  for  fifteen  minutes  to  the  passage  of  streaming  steam  or 
to  some  temperature  judged  to  be  sufficiently  high,  so  that  the  adult 
micro-organisms  contained  in  it  are  killed.  As  the  spores  remain 
uninjured,  the  medium  is  stood  aside  in  a  cool  place  for  twenty-four 
hours,  and  the  spores  allowed  slowly  to  develop  into  adult  organisms. 

When  the  twenty-four  hours  have  passed,  the  medium  is  again 


Sterilization  in  the  Autoclave 


171 


exposed  to  the  same  temperature  until  these  newly  developed 
bacteria  are  also  killed.  Eventually,  the  process  is  repeated  a 
third  time,  lest  a  few  spores  remain  alive.  When  properly  sterilized 
in  this  way  culture-media  will  remain  free  from  contamination 
indefinitely. 

A  prolonged  single  exposure  to  lower  temperatures  (6o°-7o°C.), 
known  as  pasteurization,  is  employed  for  the  destruction  of  bacteria 
in  milk  and  other  fluids  that  are  injured  or  coagulated  by  exposure 


Fig.  38. — Arnold's  steam  sterilizer  (Boston  Board  of  Health  form). 

to  ioo°C.  It  is  appropriate  only  when  the  organisms  to  be  killed 
are  without  spores  and  without  marked  resisting  powers. 

Sterilization  in  the  Autoclave. — If  it  should  be  desirable  to  sterilize 
a  medium  at  once,  not  waiting  the  three  days  required  by  the  inter- 
mittent method,  it  may  be  done  by  superheated  steam  under 
pressure,  sufficient  heat  being  generated  to  immediately  destroy 
the  spores. 

Because  of  its  convenience  many  laboratory  workers  habitually 
use  the  autoclave  for  the  sterilization  of  all  media  not  injured  by 
the  high  temperature.  The  sterilization,  to  be  complete,  requires 
that  the  exposure  shall  be  for  fifteen  minutes  at  no°C.  (six  pounds' 
pressure). 

The  media  to  be  sterilized  should  be  placed  in  the  autoclave,  the  top  firmly 
screwed  down,  but  the  escape-valve  allowed  to  remain  open  until  steam  is  freely 
generated  within  and  replaces  the  hot  air.  The  valve  is  then  closed,  and  the 
temperature  maintained  for  fifteen  minutes  or  longer  if  the  media  be  in  bulk  in 
flasks.  The  apparatus  should  be  permitted  to  cool  before  the  valve  is  opened, 


172 


Sterilization  and  Disinfection 


and  the  vacuum  be  slowly  relieved.  If  the  valve  be  opened  suddenly  the  fluids 
boil  rapidly  and  the  cotton  plugs  may  be  forced  into  the  tubes  or  flasks  by  the 
air  pressure.  The  chief  objection  to  the  use  of  the  autoclave  is  that  the  high 
temperature  sometimes  brings  about  chemic  changes  in  the  media  by  which  the 
reaction  is  altered. 

Sterilization  by  Filtration. — Liquids  that  cannot  be  subjected 
to  heat  without  the  loss  of  their  most  important  qualities  may  be 
sterilized  by  nitration — i.e.,  by  passing  them  through  unglazed 
porcelain  or  some  other  material  whose  interstices  are  sufficiently 
fine  to  resist  the  passage  of  bacteria.  This  method  is  largely 
employed  for  the  sterilization  of  the  unstable  bacterial  toxins  that 
are  destroyed  by  heat.  Various  substances  have  been  used  for 
filtration,  as  diatomaceous  earth  (Berkefeld  filters),  stone,  sand, 
powdered  glass,  etc.,  but  experimentation 
has  shown  unglazed  porcelain  to  be  the 
only  reliable  filtering  material  by  which 
to  remove  bacteria.  Even  this  material, 
whose  interstices  are  so  small  as  to  allow 
the  liquid  to  pass  through  with  great 
slowness,  is  only  certain  in  its  action  for  a 
time,  for  after  it  has  been  repeatedly  used 
the  bacteria  seem  able  to  work  their  way 
through.  To  be  certain  of  the  efficacy  of 
any  filter,  the  fluid  first  passed  through 
must  be  tested  by  cultivation  methods  to 
prove  that  all  the  bacteria  have  been 
removed. 

The  porcelain  bougies  as  well  as  their 
attachments  must  be  thoroughly  sterilized 
before  use. 

After  having  been  used,  a  porcelain 
filter  must  be  disinfected,  scrubbed,  dried 
thoroughly,  and  then  heated  in  a  Bunsen 
burner  or  blowpipe  flame  until  all  the 
organic  matter  is  consumed.  In  this  firing 
process  the  filter  first  turns  black  as  the 
organic  matter  chars,  then  becomes  white 
again  as  it  is  consumed.  The  porcelain 
must  be  dry  before  entering  the  fire,  or  it  is  apt  to  crack. 

It  should  not  be  forgotten  that  the  filtrate  must  be  received  in 
sterile  receivers  and  handled  with  care  to  prevent  subsequent 
contamination. 

The  filtration  of  water,  peptone  solution,  and  bouillon  is  com- 
paratively easy,  but  gelatin  and  blood-serum  pass  through  with 
great  difficulty,  and  speedily  gum  the  filter. 

III.  The  Disinfection  of  Instruments,  Ligatures,  Sutures,  the 
Hands,  etc. — There  are  certain  objects  used  by  the  surgeon  that 
cannot  well  be  rendered  incandescent,  exposed  to  dry  heat  at  i5o°C., 


Fig.  39. — Modern 
autoclave. 


Disinfection  of  Instruments,  etc. 


or  steamed  continuously,  or  intermittently  heated  without  injury. 
For  these  objects  disinfection  must  be  practised.  Ever  since  Sir 
Joseph  Lister  introduced  antisepsis,  or  disinfection,  into  surgery 
there  has  been  a  struggle  for  the  supremacy  of  this  or  that  highly 
recommended  germicidal  substance,  with  two  results — viz.,  that 
a  great  number  of  feeble  germicides  have  been  discovered,  and 
that  belief  in  the  efficacy  of  all  germicides  has  been  somewhat  shaken ; 


Fig.  40. — Pasteur-Chamberland   filter   arranged    to  filter  under  pressure. 

hence  the  aseptic  surgery  of  the  present  day,  which  strives  to  prevent 
the  entrance  of  germs  into  the  wound  rather  than  their  destruction  after 
admission  to  it. 

For  a  complete  discussion  of  the  subject  of  antiseptics  in  relation 
to  surgery  the  reader  must  be  referred  to  text-books  of  surgery. 


174  Sterilization  and  Disinfection 

The  Disinfection  of  the  Hands,  etc. — The  disinfection  of  the 
skin — both  the  hands  of  the  surgeon  and  the  part  about  to  be  incised 
— is  a  matter  of  the  utmost  importance.  Washing  the  hands  with 
soap,  which  has  marked  germicidal  properties,  will  in  many  cases 
suffice  to  destroy  or  remove  bacteria  from  smooth  skins.  This 
method,  which  is  regarded  by  some  surgeons  as  adequate,  is  not, 
however,  commonly  regarded  as  sufficient  protection  to  the  patient 
who  might  be  infected  by  any  remaining  micro-organisms.  To 
overcome  this,  many  surgeons  prefer  the  use  of  sterilized  gloves 


a  b  c  d 

Fig.  41. — Different  types  of  bacteriologic  filters:    a,  Kitasato;  b,  Berkefeld;  c, 
Chamberland;  d,  Reichel. 

of  thin  rubber  to  all  other  means  of  preventing  manual  infections. 
Others  prefer  to  use  detergent  and  disinfectant  measures.  The 
method  at  present  generally  employed,  and  recommended  by 
Welch  and  Hunter  Robb,  is  as  follows: 

The  nails  must  be  trimmed  short  and  perfectly  cleansed.  The  hands  are 
washed  thoroughly  for  ten  minutes  in  water  of  as  high  a  temperature  as  can 
comfortably  be  borne,  soap  and  a  previously  sterilized  brush  being  freely  used, 
and  afterward  the  excess  of  soap  washed  off  in  clean  hot  water.  The  hands  are 
then  immersed  for  from  one  to  two  minutes  in  a  warm  saturated  solution  of 
permanganate  of  potassium,  then  in  a  warm  saturated  solution  of  oxalic  acid, 
until  complete  decolorization  of  the  permanganate  occurs,  after  which  they  are 
washed  free  from  the  acid  in  clean  warm  water  or  salt  solution.  Finally,  they 
are  soaked  for  two  minutes  in  a  i  :  500  solution  of  bichlorid  of  mercury. 

Lock  wood,*  of  St.  Bartholomew's  Hospital,  recommends,  after  the  use  of  the 
scissors  and  penknife,  scrubbing  the  hands  and  arms  for  three  minutes  in  hot 
water  and  soap  to  remove  all  grease  and  dirt.  The  scrubbing  brush  ought  to  be 
steamed  or  boiled  before  use,  and  kept  in  i  :  1000  biniodid  of  mercury  solution. 
When  the  soapsuds  have  been  thoroughly  washed  away  with  plenty  of  clean 
water,  the  hands  and  arms  are  thoroughly  washed  and  soaked  for  not  less  than 

*  "Brit.  Med.  Jour.,"  July  n,  1896. 


Disinfection  of  Sick-chambers,  etc.  175 

two  minutes  in  a  solution  of  biniodid  of  mercury  in  methylated  spirit;  i  part  of 
the  biniodid  in  500  of  the  spirit.  Hands  that  cannot  bear  i  :  1000  bichlorid  and 
5  per  cent,  carbolic  solutions  bear  frequent  treatment  with  the  biniodid.  After 
the  spirit  and  biniodid  have  been  used  for  not  less  than  two  minutes,  the  solution 
is  washed  off  in  i  :  2000  or  i  :  4000  biniodid  of  mercury  solution. 

It  is  a  mistake  to  insist  upon  the  employment  of  disinfecting 
solutions  of  a  strength  injurious  to  the  skin.  It  must  be  obvious 
to  every  one  that  rough  skins  with  numerous  hang-nails  and  fissures 
offer  greater  difficulties  to  be  overcome  in  disinfection,  and  more 
readily  convey  micro-organisms  into  the  wound  than  smooth, 
soft  skins. 

Sterilization  of  Ligatures,  etc. — Catgut  cannot  be  sterilized  by 
boiling  without  deterioration.  The  present  method  of  treatment  is 
to  dry  it  in  a  hot-air  chamber  and  then  boil  it  in  cumol,  which  is 
afterward  evaporated  and  the  skeins  preserved  in  sterile  test-tubes 
or  special  receptacles  plugged  with  sterile  cotton.  Cumol  was  first 
introduced  for  this  purpose  by  Kronig,  as  its  boiling-point  is  168°— 
i78°C.,  and  thus  sufficiently  high  to  kill  spores.  The  use  of  cumol 
for  the  sterilization  of  catgut  has  been  carefully  investigated  by 
Clarke  and  Miller.* 

Catgut  may  also  and  equally  well  be  sterilized  by  the  use  of 
chemical  agents.  This  subject  has  been  carefully  reviewed  by  Ber- 
tarelli  and  Bocchia,f  who  regard  the  method  of  Claudius  and 
the  modification  of  it  by  Rogone  as  the  best.  The  method  of 
Claudius  is  to  roll  the  catgut  into  skeins  and,  without  taking  any 
precautions  to  remove  any  fat  it  may  contain,  place  it  in  a  mixture 
of  iodin  i,  iodid  of  potassium  i,  and  distilled  water  100.  After 
immersion  for  eight  days  the  catgut  is  removed,  under  aseptic  pre- 
cautions, to  alcohol  or  to  3  per  cent,  carbolic  solution,  in  which  it  is 
indefinitely  preserved  for  use. 

Ligatures  of  silk  and  silkworm  gut  are  boiled  in  water  immediately 
before  using,  or  are  steamed  with  the  dressings,  or  placed  in  test- 
tubes  plugged  with  cotton  and  steamed  in  the  sterilizer. 

Sterilization  of  Surgical  Instruments,  etc. — In  most  hospitals 
instruments  are  boiled,  before  using,  in  a  i  to  2  per  cent,  soda  (sodium 
carbonate,  sodium  bicarbonate,  or  sodium  biborate)  solution,  as 
plain  water  has  the  disadvantage  of  rusting  them.  During  the 
operation  they  are  either  kept  in  the  boiled  water  or  in  a  carbolic 
solution,  or  are  dried  with  a  sterile  towel.  Andrews  makes  special 
mention  of  the  fact  that  the  instruments  must  be  completely 
immersed  to  prevent  rusting. 

Disinfection  of  the  Wound. — Cleansing  solutions  (normal  salt 
solution)  and  disinfecting  solutions  (such  as  i :  10,000  to  i  :  1000 
bichlorid  of  mercury)  are  only  applied  to  septic  wounds. 

IV.  The  Disinfection  of  Sick-chambers,  Dejecta,  etc. — The 
Air  of  the  Sick-room. — It  is  impossible  to  sterilize  or  disinfect  the 

*  "Bull,  of  the  Johns  Hopkins  Hospital,"  Feb.  and  March,  1896. 
f  "Centralbl.  fur  Bakt.  u.  Parasitenk.,"  Orig.  L,  620. 


176  Sterilization  and  Disinfection 

atmosphere  of  a  room  during  its  occupancy  by  the  patient.  It  is 
entirely  useless  to  place  beneath  the  bed  or  in  the  corner  of  a  room 
small  receptacles  filled  with  carbolic  acid  or  chlorinated  lime.  These 
can  serve  no  purpose  for  good,  and  may  do  harm  by  obscuring  odors 
emanating  from  harmful  materials  that  should  be  removed  from 
the  room.  The  practice  is  only  comparable  to  the  old  faith  in  the 
virtue  of  asaf etida  tied  in  a  corner  of  the  handkerchief  as  a  preventive 
of  cholera  and  smallpox. 

DISINFECTANTS 

Before  one  is  able  to  make  a  scientific  application  of  any  germicidal 
substance  it  is  necessary  to  become  acquainted  with  its  micro- 
organism-destroying powers.  This  may  seem  at  first  thought  to 
be  a  simple  matter,  but  is,  in  reality,  one  of  great  complexity  and 
difficulty,  for  the  various  micro-organisms  show  marked  variations 
in  their  powers  of  endurance;  different  stages  in  the  development  of 
the  micro-organisms  show  different  degrees  of  resisting  power,  and 
the  conditions  under  which  the  germicide  meets  the  micro-organism 
effect  marked  variations  in  action.  These  factors  make  it  necessary 
to  vary  the  process  of  disinfection  according  to  the  exact  purpose 
to  be  achieved. 

Let  two  examples  serve  to  illustrate  these  requirements :  Bichlorid 
of  mercury  is  one  of  the  most  powerful,  reliable,  and  generally  useful 
germicides,  but  the  strength  of  its  solutions  must  vary  according 
to  the  purpose  for  which  they  are  intended.  It  kills  cocci  and  non- 
sporogenic  bacilli  in  dilutions  of  i :  10,000  in  .from  five  minutes  to 
twenty-four  hours,  but  to  kill  anthrax  spores  requires  twenty-four 
hours'  immersion  in  i :  2000  solution.  If  albuminous  substances  are 
present  in  the  medium  containing  the  micro-organisms  they  precipi- 
tate the  salt  immediately,  diminishing  the  strength  of  the  solution 
and  so  retarding  or  perhaps  preventing  the  germicidal  action.  Again, 
certain  micro-organisms  are  defended  from  the  action  of  destructive 
agents,  and  among  them  the  germicides,  through  the  presence  of 
waxy  matter  in  their  substance.  Such  is  the  case  with  the  acid-fast 
organisms,  and  notably  the  tubercle  bacillus.  Antiformin,  a  com- 
bination composed  of  equal  parts  of  liquor  sodas  chlorinatae  and  a 
15  per  cent,  solution  of  caustic  soda,  immediately  dissolves  the 
great  majority  of  micro-organisms,  but  has  no  destructive  action 
upon  the  tubercle  bacillus. 

The  most  useful  germicidal  substances  act  destructively  upon  the 
micro-organisms  by  forming  chemical  compounds  with  their  cyto- 
plasm. Thus,  the  salts  of  mercury  unite  with  the  protoplasm  to  form 
an  albuminate  of  mercury.  Other  germicidal  agents  dissolve  or 
coagulate  the  protoplasm;  still  others  oxidize  and  so  completely 
destroy  the  cells.  In  the  process  of  germicidal  action  many  and 
varied  activities  are  at  work,  and,  as  all  are  not  understood,  the 
subject  is  a  difficult  one  to  handle  in  a  limited  amount  of  space. 


Inorganic  Disinfectants  177 

With  the  salts,  acids,  and  bases  it  appears  from  the  researches  of 
Kronig  and  Paul*  that  ionization  in  solution  plays  an  important 
part  in  the  destruction  of  micro-organisms.  They  found  that 
double  metallic  salts,  in  which  the  metal  is  a  constituent  of  a  com- 
plex ion  in  which  the  concentration  of  the  dissociated  metal  ions  is 
consequently  very  low,  have  very  little  germicidal  power,  but  that 
simple  salts,  in  which  the  condition  is  reversed,  have  correspondingly 
higher  germicidal  power.  Dissociation,  therefore,  seems  to  have 
much  to  do  with  the  matter. 

Inorganic  Disinfectants. 

ACIDS. — These  agents  are  seldom  employed,  since  the  concentration  required 

makes  them  objectionable. 

ALKALIS. — The  same  holds  good  with  regard  to  these  agents. 
SALTS. — In  this  group  we  find  some  of  the  most  powerful  and  most  useful 

germicidal  substances. 

Copper  Sulphate. — It  is  curious  and  interesting  that  while  this  salt  is  highly 
destructive  to  algae  and  other  low  forms  of  vegetable  life,  it  is  not  of 
much  value  for  the  destruction  of  bacteria.  Its  chief  use  is  for  the 
destruction  of  the  green  alga?  that  sometimes  render  the  water  of 
reservoirs  dirty  and  offensive.  Some  of  the  salt  contained  in  a  gunny- 
sack  and  permitted  to  drag  to  and  fro  over  the  surface  of  the  water 
behind  a  slowly  rowed  boat  usually  accomplishes  the  end,  the  actual 
quantity  dissolving  in  the  water  being  almost  infinitesimal. 
Mercuric  Chlorid  (HgC^). — This  is  probably  the  most  generally  useful 

as  well  as  one  of  the  strongest  germicides. 

A  study  of  its  activity  under  varying  conditions  is  instructive  as  exemplifying 
the  varying  behavior  of  germicides  under  the  varying  conditions  under  which 
they  may  be  employed. 

First,  it  makes  great  difference  whether  the  mercuric  chlorid  is  added  to  the 
substratum  containing  the  bacteria,  or  whether  the  bacteria  are  added  to  solu- 
tions of  the  germicide. 

Thus,  when  the  salt  is  dissolved  in  gelatin  in  a  concentration  of  i  :  1,000,000, 
anthrax  bacilli  cannot  grow.  If  it  is  dissolved  in  blood-serum,  the  concentration 
must  be  increased  to  i  :  10,000  to  prevent  their  growth. 

When  the  anthrax  spores  are  dropped  in  solutions  of  the  salt,  Kronig  and  Paul 
found  that  they  were  killed  in  twelve  to  fourteen  minutes  by  1 165  solutions;  in 
eighty  minutes  by  i  1500  solutions,  and  in  two  hours  by  1:1000  solutions. 
When  the  reaction  takes  place  in  albuminous  media  Behring  and  Nochtf  found 
that  much  more  time  was  required.  Thus,  the  destruction  of  the  spores  by  a 
i :  200  solution  required  eighty  minutes,  and  a  i  :  1000  solution  twenty-four 
hours  to  completely  kill  all  of  the  spores. 

Laplace  |  and  Panfili§  found  that  the  addition  of  5  per  cent,  of  tartaric  or 
hydrochloric  acid  facilitated  the  germicidal  action  through  the  prevention  of 
albuminate  of  mercury  formation.  Liibbert  and  Schneider  and  Behring  have 
used  sodium  chlorid  and  ammonium  chlorid.  Both  of  these  salts  diminish  the 
germicidal  action  of  the  mercuric  salt  about  one-half.  Notwithstanding  this, 
however,  the  "antiseptic  tablets"  in  common  use  for  surgical  and  household 
purposes  contain  one  or  both  of  these  salts,  added  for  the  purpose  of  preventing 
the  precipitation  of  the  mercuric  compounds  formed  in  the  presence  of  alkaline 
albuminous  materials,  such  as  blood,  pus,  sputum,  feces,  etc. 

The  addition  of  about  25  per  cent,  of  alcohol  to  the  solution  of  the  mercuric 
salt  greatly  enhances  its  value.  Strong  alcoholic  solutions  are,  however,  less 
useful  than  aqueous  solutions,  for  the  95  or  100  per  cent.'  alcohol  dehydrates  the 
micro-organisms  and  prevents  the  diffusion  currents  by  which  the  mercury  is 
carried  into  their  substance. 

*  "Zeitschrift  fur  Hygiene,"  1897,  xxv,  i. 
t  Ibid.,  rx,  432. 

'Deutsche  med.  Wochenschrift,"  1887,  866;  1888,  121. 

'Ann.  Ig.  Roma,"  1893,  in,  527. 


178  Sterilization  and  Disinfection 

For   most   purposes   a    i  :  2000   solution    of    the    mercuric   chlorid   is  to  be 
recommended. 

Silver  Nitrate  (AgNO3). — The  solutions  of  this  salt  are  probably  more 
useful  than  the  frequency  of  their  employment  might  suggest.  They 
have,  however,  the  disadvantages  of  decomposing  when  kept  in  the 
light  and  of  making  black  stains  when  applied  in  concentrated  form 
to  the  skin  or  dressings. 

The  germicidal  power  of  the  salt  in  aqueous  solution  is  less  than  that 
of  the  mercuric  chlorid,  but  the  power  in  albuminous  fluids  is  greater. 
Anthrax  spores  in  blood-serum  are  killed  in  seventy  hours  in  a  i  :  1 2,000 
solution.  The  addition  of  other  salts,  as  ammonium  salts,  interferes 
with  the  germicidal  activity  by  inhibiting  ionization. 

Combinations  of  the  silver  nitrate  with  albuminous  compounds,  and 
variously  known  as  argonin,  argentum  casein,  argyrol,  protargol,  etc., 
have  been  used  where  the  disinfecting  power  of  the  silver  is  sought  for 
with  the  least  amount  of  irritation  and  the  deepest  degree  of  pene- 
tration, as  in  the  treatment  of  gonorrhea. 

Potassium  Permanganate  (KMnO^. — Solutions  of  this  salt  seem  to  act  by 
virtue  of  a  strong  oxidizing  power.  In  2  per  cent,  solutions  anthrax 
spores  are  killed  in  forty  minutes;  in  4  per  cent,  solutions,  within 
fifteen  minutes.  Koch's  experiments  showed  less  activity  of  the 
germicidal  power  against  anthrax  spores.  In  his  hands  a  5  per  cent, 
solution  seemed  to  require  about  a  day  to  effect  complete  destruction. 
A  i  per  cent,  solution  kills  the  pus  cocci  in  ten  minutes;  a  i  :  10,000 
solution  kills  plague  bacilli  in  five  minutes. 

The  chief  difficulty  is  that  the  salt  is  quickly  reduced  and  its  strength 
destroyed  by  the  organic  substrata  in  which  the  bateria  are  contained. 
HALOGENS  AND  COMPOUNDS. — Those  with  the  lowest  atomic  weight  have 
the  greatest  disinfecting  power. 

Chlorin. — This  is  usually  employed  in  the  form  of  chlorinated  lime.  It 
seems  to  be  a  mixture  of  calcium  hypochlorite,  Ca(ClO2),  and  calcium 
chlorid,  CaOCl2.  The  addition  of  any  acid,  including  the  atmospheric 
CO2,  causes  the  evolution  of  Cl.  The  powder  is  readily  soluble  and 
solutions  of  i  '.500  kill  vegetative  forms  of  most  bacteria  in  a  few 
minutes  (not,  however,  resisting  spores). 

A  proprietary  compound  known  as  "  electrozone,"  made  by  electro- 
lyzing  sea-water  in  such  a  manner  that  magnesia  and  chlorin  are 
liberated  and  magnesium  hypochlorite  and  magnesium  chlorid  formed, 
is  a  cheap  and  useful  chlorin  disinfectant.  Nissen  found  that  1.5  per 
cent,  of  it  killed  typhoid  bacilli  in  a  few  minutes;  Rideal,  that  i  1400 
to  500  dilutions  of  it  disinfected  sewage  in  fifteen  minutes;  and  Delepine, 
that  i  150  (equal  to  0.66  per  cent,  of  chlorin)  rapidly  killed  the  tubercle 
bacillus  and  i  :io  (equal  to  3.3  per  cent,  chlorin)  killed  anthrax  spores. 

lodin  Terchlorid  (Ids). — This  compound,  which  is  so  unstable  that  it 
only  keeps  in  an  atmosphere  of  Cl-gas,  has  great  germicidal  action, 
that  probably  depends  upon  the  readiness  with  which  it  decomposes. 
In  solutions  of  i  :  1000  it  kills  vegetative  bacteria  in  a  few  minutes, 
and  in  i  :  100  it  kills  anthrax  spores  with  equal  rapidity.  The  presence 
of  organic  and  albuminous  materials  does  not  interfere  with  the  germi- 
cidal action. 

Organic  Disinfectants. 

Carbolic  Acid  (CeHsOH)  is  the  most  important  and  generally  useful  of 
these.  It  has  the  advantage  of  being  cheap  and  easily  kept  and 
handled.  In  the  pure  state  it  consists  of  colorless  acicular  crystals. 
When  exposed  to  the  atmosphere  it  takes  up  water  and  gradually 
becomes  a  brownish-yellow  oily  fluid.  The  crystals  and  deliquesced 
crystals  have  powerful  escharotic  properties  and  cannot  be  touched 
without  destruction  of  the  skin.  In  2  to  3  or  5  per  cent,  solutions 
carbolic  acid  destroys  most  bacteria  within  a  few  minutes.  Anthrax 
and  other  powerfully  resisting  spores,  however,  require  prolonged 
exposure.  Tetanus  spores  are  said  not  to  be  killed  in  less  than  fifteen 
hours.  There  is  no  ionization;  the  reagent  seems  to  act  by  coagulating 
the  bacterial  protoplasm. 


Organic  Disinfectants  179 

Though  carbolic  acid  has  been  for  a  quarter  of  a  century  a  favorite 
surgical  disinfectant,  the  application  of  5  per  cent,  solution  to  the  skin 
has  so  frequently  caused  gangrene  that  it  is  at  present  in  some  merited 
disfavor. 

Closely  related  to  carbolic  acid  and  other  products  of  coal-tar  dis- 
tillation are  orthocresol,  metacresol,  and  paracresol.  "Trikresol,"  a 
much  used  antiseptic,  is  a  commercial  product  consisting  of  a  mixture 
of  all  three  of  the  cresols.  It  is  more  strongly  germicidal  than  carbolic 
acid,  but  is  less  soluble  in  water.  It  is  or  has  been  largely  used  for 
addition  to  therapeutic  serums  in  the  proportion  of  0.4  per  cent,  as  an 
antiseptic.  Such  addition  causes  the  formation  of  an  albuminous  pre- 
cipitate in  which,  doubtless,  much  of  the  antiseptic  is  lost,  for  upon  its 
removal  or  even  upon  its  sedimentation  resisting  forms  of  bacteria  may 
grow  in  the  serum.  It  cannot,  therefore,  be  looked  upon  as  a  reliable 
preservative. 

"Lysol"  is  said  to  be  a  solution  of  coal-tar  cresol  in  potassium  soap. 
It  has  the  advantage  of  forming  a  lather-like  soap,  so  that  it  can 
be  employed  both  as  a  cleanser  and  disinfectant.  In  i  per  cent,  solu- 
tions it  is  capable  of  destroying  cocci,  typhoid  bacilli,  and  other  micro- 
organisms of  low  resisting  power. 

"Creolin"  is  also  a  combination  of  cresols  with  potassium  soap. 
When  added  to  water  it  immediately  forms  an  emulsion.  It  has  been 
much  used  in  obstetric  practice,  where  it  has  earned  more  reputation 
than  it  deserves. 

"Formalin." — This  is  Schering's  commercial  denomination  of  a  30  to  40 
per  cent,  aqueous  solution  of  formaldehyd  gas  (H — COH)  or  formic 
aldehyd.  The  solution  is  highly  germicidal  so  long  as  it  is  fresh.  When 
exposed  for  long  to  the  atmosphere  it  polymerizes  into  trioxymethylene 
and  paraformaldehydeand  greatly  loses  its  power.  A  10  per  cent,  solu- 
tion of  formalin  kills  pus  cocci  in  half  an  hour.  A  5  per  cent,  solution 
kills  cholera spirilli  in  three  minutes;  anthrax  bacilli,  in  fifteen  minutes; 
anthrax  spores,  in  five  hours.  Pure  formalin  kills  anthrax  spores  in 
ten  to  thirty  minutes.  Strong  solutions  are  extremely  irritating  and 
so  not  applicable  in  surgery.  They  are,  however,  of  great  use  for 
household  disinfection.  Formalin  and  formaldehyd  gas  find  their  chief 
usefulness  for  the  aerial  disinfection  of  sick  chambers  and  domiciles, 
where  they  are  either  used  as  a  spray  or  the  gas  evolved  by  chemical 
means  or  by  heat,  as  will  be  shown  below. 

Peroxid  of  hydrogen  (H2O2)  is  germicidal  through  its  power  to  liberate 
the  nascent  O.  It  quickly  decomposes  when  brought  into  contact 
with  organic  matter,  and,  therefore,  has  a  very  limited  sphere  of 
usefulness. 

The  following  tables,  compiled  by  Hiss  from  Fliigge,  will  show 
the  comparative  values  of  the  commonly  employed  antiseptics  and 
germicides : 

Certain  fundamental  principles  govern  the  rationale  of  disin- 
fection, and  must  be  kept  in  mind:  (i)  the  reagent  employed  should 
be  known  to  act  destructively  upon  bacteria;  (2)  it  must  be  applied 
to  the  bacteria  to  be  killed;  (3)  it  must  be  applied  in  sufficiently 
concentrated  form,  and  (4)  it  must  be  left  in  contact  with  the 
bacteria  long  enough  to  accomplish  the  effect  desired. 

During  the  period  of  illness  the  chamber  in  which  the  patient -is 
confined  should  be  freely  ventilated.  An  abundance  of  fresh,  pure 
air  is  a  comfort  to  the  patient  and  a  protection  to  the  doctor  and 
nurse. 

After  recovery  or  death  one  should  rely  less  upon  fumigation 
than  upon  disinfection  of  the  walls  and  floor,  the  similar  disinfection 
of  the  wooden  part  of  the  furniture,  and  the  sterilization  of  all  else. 


i8o 


Sterilization  and  Disinfection 


The  fumes  of  sulphur  do  some  good,  especially  when  combined  with 
steam,  but  are  greatly  overestimated  in  action  and  are  very  destructive 
to  furnishings,  so  that  they  are  rapidly  giving  way  to  the  more 
satisfactory,  less  destructive,  and  equally  germicidal  formaldehyd 
vapor. 

INHIBITION  STRENGTHS  OF  VARIOUS  ANTISEPTICS 

(Adapted  from  Fliigge,  Leipzig,  1902) 


Anthrax  Bacilli 

Other  Bacteria 

Putrefactive 
Bacteria  in 
Bouillon 

ACIDS 
Sulphuric 

1  1  3000 

Choi,  spir   i  :  6000 

Hydrochloric           

1  :  3000 

B.  diph.  i  :  3000 

Sulphurous.              

B.  mallei  i:  700 
B.  typh.  1:500 
Choi.  spir.  i  :  1000 

I    6000 

Arsenous  

I     2OO 

Boric  
ALKALIES 
Potass,  hydrox  

1:800 

i  :  700 

B.diph.  i  :  600 

I    IOO 

Arnmon  hydrox 

I  *  7OO 

Choi.  spir.  i  :  400 
B.  typh.  1:400 
Choi   spir   i  :  500 

Calcium  hydrox 

B.  typh.  1:500 
Choi  spir   I:TIOO 

SALTS 
Copper  sulphate 

B.  typh.  i:  noo 

I    IOOO 

Ferric  sulphate             .    .    . 

I    90 

Mercuric  chlorid 

I    100,000 

B.  typh.  1:60,000 

I    20,000 

Silver  nitrate  .          

I    60,000 

Choi,  spir., 

Potass,  perman  

I     IOOO 

B.  typhosus  i  :  50,000 

I    500 

HALOGENS  AND  COMPOUNDS 
Chlorin  

I    1500 

I  4000 

Bromin 

I    1500 

I     2OOO 

lodin 

I    5000 

I    5OO*O 

Potass  iodid 

i   7 

Sodium  chlor  

I  60 

ORGANIC  COMPOUNDS 
Ethyl  alcohol 

I    12 

I    IO- 

Acetic  and  oxalic  acids 

B.  diph.  1:500 

i  40 

Carbolic  acid 

I    800 

B.typh.  i  :4oo 

Benzoic  acid.  .  .  . 

I     IOOO 

Choi.  spir.  1:600 

Salicylic  acid  
Formalin  (40%  formaldehyd) 

I  1500 

Choi.  spir.  i  :  20,000 

i  :  1000 

Camphor.  . 

I     IOOO 

Staphylo.  1:5000 

Thymol 

I     IO  OOO 

i  :  3  500 

Oil  mentha  pip  

I    3000 

Oil  of  terebinth  
Peroxid  of  hydrogen 

I  8000 

i  :  2000 

Formaldehyd  is  probably  the  best  germicide  that  has  yet  been 
recommended.     Its  use  for  the  disinfection  of  rooms  and  hospital 
wards  was  first  suggested  by  Trillat*  in  1892,  but  it  did  not  make 
*  "Compte  rendu  de  1'Acad.  des  Sciences,"  Paris,  1892. 


Comparison  of  Disinfectants 


181 


much  stir  in  the  medical  world  until  a  year  or  more  had  passed  and 
a  40  per  cent,  solution  of  the  gas,  under  the  name  of  "Formalin," 
had  been  placed  upon  the  market.  Care  must  be  exercised  in 
handling  the  fluid,  that  the  hands  do  not  become  wet  with  it,  as  it 
hardens  the  skin  and  deadens  sensation.  The  vapor  is  exceedingly 
irritating  to  the  mucous  membrane  of  the  eyes  and  nose. 

BACTERICIDAL  STRENGTH  OF  COMMON  DISINFECTANTS 

(Adapted  from  Fliigge,  Leipzig,  1902) 


Streptococci 
and  Sta- 
phylococci 

Anthrax  and  Typhoid  Bacilli, 
Cholera  Spirillum 

Anthrax  Spores 

5  minutes 

5  minutes 

2  to  24  hours 

ACIDS 
Sulphuric  
Hydrochloric  
Sulphurous 

i:  10 
i:  10 

t:  100 
1:100 

i:i500 
1:1500 

Typhoid 
i:  700 
1:300  (Gas 
10  vol.  %) 
1:30 

1:50  in  10  days 
1:50  in  10  days. 

Cone.    sol.     in- 
complete disin- 
fection. 

i  :  20  (5  days) 
i:  2000(26  hrs.) 

i:  20  (i  day) 
i:  20  (i  hr.) 

2%  (in  i  hr.) 
1:1000    (in     12 
hours) 

Alcol.  50%  for  4 
months     with- 
out kill  ing 
spores  (Koch*) 
1:20    (4    to    45 
days)    (at    40° 
in  3  hours) 

(10%  in  5  hrs.) 
1:20  (in  6  hrs.) 

i:  100  (in  i  hr.) 
3:100  (in  i  hr.) 

Sulphurous 

Boric     .    .        

ALKALIES 
Potass,  hydrox.  .  .  . 
Arnmon   hydrox 

i  :5 

i  1300 
1:300 
i:  1000 

Calcium 

SALTS 
Copper  sulphate 

Mercuric  chlor  
Silver  nitrate 

i  :  10,000  to 
.1000 
i:  1000 
i:  200 

i  :  2000 

i  :  10,000 
i  :  4000 

Potass,  permang.  .  .  . 
"Calc.  chlorid."..  .  . 
HALOGENS  AND  COM- 
POUNDS 
Chlorin 

i  :  500 

i% 
i  :  1000 

70%—  10 
minutes 

Cholera 
i  :  200 
Typh.  1:50 
1:300 
i  :  100 

1:20 
i:  200 

i% 
i:  200 

70%—  15 
minutes 

1:60 

i  :  300 

i:  1000 
1:10 

Cone. 

Trichlorid  of  iodin.. 

ORGANIC  COMPOUNDS 
Ethyl  alcohol  

Acetic  and  oxalic  acids. 
Carbolic  acid 

i  :  200  to  300 
1:300 

i  :  3000 
i:  1000 
1:500 

Lysol  

Creolin  
Salicylic  acid  
Formalin  (40%  for- 
maldehyd). 
Peroxid  of  hydrogen 

The  solution  can  be  employed  to  spray  the  walls  and  floors  of 
*  Koch,  Arb.  a.  d.  kais.  Gesundheitsamt,  1881,  i. 


1 82  Sterilization  and  Disinfection 

rooms,  though  Rosenau*  finds  that  unless  the  spray  discharged 
from  a  large  atomizer  be  very  fine,  its  action  is  uncertain. 

The  original  method  of  disinfection,  suggested  by  Robinson,  f 
consisted  of  the  evolution  of  the  gas  by  volatilizing  methyl  alcohol, 
and  passing  the  vapor  over  heated  asbestos.  Shortly  many  efficient 
forms  of  apparatus  were  placed  upon  the  market,  for  the  evolution 
of  the  gas  or  for  discharging  it  from  the  solution. 

It  is  not  necessary  to  use  a  special  apparatus  in  order  to  disinfect 
with  formaldehyd;  one  can,  in  an  emergency,  hang  up  a  number 
of  sheets,  saturated  with  the  40  per  cent,  solution,  in  the  room  to 
be  disinfected.  The  number  of  sheets  must  vary  with  the  size  of 
the  room,  as  each  is  able  to  evolve  but  a  certain  amount  of  the 
gas,  and  the  quantity  necessary  for  disinfection  varies  with  the  size 
of  the  room. 

One  of  the  best  methods  of  evolving  the  gas  for  purposes  of  dis- 
infection is  that  devised  by  Evans  and  Russell  |  who  combine  the 
40  per  cent,  solution  of  formaldehyd  with  permanganate  of  po- 
tassium, when  an  almost  explosive  liberation  of  the  gas  takes  place. 

Frankforterf  found  that  a  good  method  of  escaping  the  un- 
desirable features  of  the  gaseous  evolution  was  to  mix  the  powder 
of  permanganate  of  potassium  with  an  equal  volume  of  sand,  so 
that  the  formaldehyd  solution  is  brought  more  slowly  into  contact 
with  the  permanganate,  under  conditions  unfavorable  to  the  forma- 
tion of  oxids  of  manganese,  such  as  otherwise  tend  to  coat  the  grains 
of  permanganate  and  prevent  further  reaction  between  the  formal- 
dehyd solution  and  the  permanganate. 

The  employment  of  calcium  carbide  for  the  same  purpose  is 
suggested  by  Evans.§  The  best  results  were  obtained  when  the 
calcium  carbide  was  in  lumps  about  the  size  of  a  pea;  when  the  formal- 
dehyd solution  was  diluted  with  an  equal  volume  of  water,  and  when 
the  diluted  formaldehyde  was  added  to  the  carbide  in  the  propor- 
tion of  5  cc.  of  the  former  to  3  grams  of  the  latter.  In  the  per- 
manganate method  the  quantity  of  formalin  (or  37-40  per  cent,  for- 
maldehyd in  water)  should  equal  200  cc.  to  1000  cubic  feet  of 
space,  but  in  the  carbide  method  500  cc.  must  be  used  to  achieve 
the  same  result.  Evans,  therefore,  prefers  the  permanganate 
method. 

To  disinfect  with  formaldehyd  or  any  gaseous  disinfectant,  the 
room  must  be  carefully  closed,  the  cracks  of  the  windows  and 
doors  being  sealed  by  pasting  strips  of  paper  over  them.  If  an 
apparatus  is  used,  it  is  set  in  action,  the  discharged  vapor  entering 
the  room  through  the  keyhole  or  some  other  convenient  aperture, 

*"  Disinfection  and  Disinfectants,"  P.  Blakiston's  Son  &  Co.,  Philadelphia, 
1902. 

f  "Ninth  Report  of  the  State  Board  of  Health  of  Maine,"  1896. 

I" Reports  and  Papers  of  the  American  Public  Health  Association,"  1906, 
vol.  xxxn,  part  n,  p.  114. 

§  Ibid.,  p.  108. 


Disinfection  of  Dejecta  183 

the  gas  being  allowed  to  act  undisturbed  for  some  hours,  after  which 
the  windows  and  doors  are  all  thrown  open  to  fresh  air  and  sunlight. 

If  sheets  are  hung  up,  or  the  permanganate  method  employed, 
the  windows  and  doors,  other  than  that  by  means  of  which  the 
operator  is  to  escape,  are  closed  and  sealed.  If  the  permanganate  of 
potassium  or  calcium  carbide  methods  are  to  be  employed,  the 
cracks  about  the  doors  and  windows  are  sealed  with  paper,  a  dish- 
pan  or  wash-tub  is  placed  in  the  center  of  the  room,  and  in  it  the 
can  containing  the  permanganate  or  carbide  and  sand.  The  for- 
maldehyd  solution  is  poured  on,  the  operator  making  his  escape, 
closing  and  sealing  the  door  behind  him.  Any  closets  in  the  room 
must  be  left  open  so  that  they  and  their  contents  may  be  disinfected 
with  the  room. 

So  far  as  is  known  at  present,  superficial  disinfection  by  formal- 
dehyd  leaves  little  to  be  desired.  Care  must,  however,  be  exercised 
to  see  that  the  required  volume  of  gas  is  generated  to  disinfect  the 
apartment.  A  sufficient  concentration  of  the  gas  is  absolutely  necessary 
and  the  method  selected  should  be  one  capable  of  discharging  the 
gas  in  a  short  time,  so  that  it  immediately  pervades  the  atmosphere. 

Disinfection  with  formaldehyd  is,  however,  only  superficial,  its 
penetrating  powers  being  limited.  The  discharge  of  gas  into  the 
room  should  only  be  preliminary  to  other  and  more  thorough  dis- 
infection and  sterilization  of  its  contents  by  the  application  of 
solutions  of  disinfectants  to  the  woodwork,  and  the  baking  of  the 
mattresses  and  pillows  and  the  boiling  of  the  linen,  etc. 

The  Dejecta. — In  diphtheria  the  expectoration  and  nasal  dis- 
charges are  highly  infectious  and  should  be  received  in  old  rags  or 
in  Japanese  paper  napkins — not  handkerchiefs  or  towels — and 


Fig.  42. — Pasteboard    cup    for   receiving   infectious    sputum.     When  used  the 
pasteboard  can  be  removed  from  the  iron  frame  and  burned. 

should  be  burned.  The  sputum  of  tuberculous  patients  should  either 
be  collected  in  a  glazed  earthen  vessel  which  can  be  subjected  to 
boiling  and  disinfection,  or,  as  is  an  excellent  plan,  should  be  re- 
ceived in  Japanese  rice-paper  napkins,  which  can  at  once  be  burned. 
These  napkins  are  not  quite  so  good  as  the  small  pasteboard  boxes 
recommended  by  some  city  boards  of  health,  because,  being  highly 
absorbent,  the  sputum  is  apt  to  soak  through  and  soil  the  fingers. 


184  Sterilization  and  Disinfection 

For  the  fastidious  patients,  cut-glass  bottles  with  tightly  fitting  lids 
are  used  to  collect  the  sputum,  and  as  these  are  not  unsightly  the 
patients  make  no  objection  to  carrying  them  about  with  them. 
Tuberculous  patients  should  be  provided  with  rice-paper  instead  of 
handkerchiefs,  and  should  have  their  napkins,  towels,  knives,  forks, 
spoons,  plates,  etc.,  kept  strictly  apart  from  the  others  of  the  house- 
hold and  carefully  sterilized  by  boiling  after  using.  Patients  with 
sensitive  dispositions  need  never  be  told  of  these  arrangements. 

The  excreta  from  cases  of  typhoid  fever  and  cholera  require 
particular  attention.  These,  and  indeed  all  alvine  matter  the  pos- 
sible source  of  infection  or  contagion,  should  be  received  in  glazed 
earthen  vessels  and  immediately  and  intimately  mixed  with  a  5 
per  cent,  solution  of  chlorinated  lime  (containing  25  per  cent,  of 
chlorin)  if  semi-solid,  or  with  the  powder  if  liquid,  and  allowed  to 
stand  for  an  hour  before  being  thrown  into  the  drain. 

Thoughtful  consideration  should  always  be  given  the  germicides 
used  to  disinfect  the  discharges,  lest  combination  of  the  chemical 
with  ingredients  of  the  discharge  produce  inert  compounds.  Thus, 
bichlorid  of  mercury  cannot  be  used  because  it  forms  an  inert 
compound  with  albumin. 

The  Clothing,  etc. — The  bed-clothing,  towels,  napkins,  handker- 
chiefs, night-robes,  underclothes,  etc.,  used  by  a  patient  suffering 
from  an  infectious  disease,  as  well  as  the  towels,  napkins,  handker- 
chiefs, caps,  aprons,  and  outside  dresses  worn  by  the  nurse,  should  be 
regarded  as  infective  and  carefully  sterilized.  The  only  satisfactory 
method  of  doing  this  is  by  prolonged  subjection  to  steam  in  a  special 
apparatus;  but,  as  this  is  only  possible  in  hospitals,  the  next  best 
thing  is  boiling  for  some  time  in  the  ordinary  wash-boiler.  In  drying, 
the  wash  should  hang  longer  than  usual  in  the  sun  and  wind.  Woolen 
underwear  can  be  treated  exactly  as  if  made  of  cotton.  The  woolen 
outer  clothing  of  the  patient,  if  infective,  requires  special  treat- 
ment. Fortunately,  the  infection  of  the  outer  garments  is  unusual. 
The  only  reliable  method  for  their  sterilization  is  prolonged  ex- 
posure to  hot  air  at  i  io°C.  In  private  practice  it  often  becomes  a 
grave  question  what  shall  be  done  with  these  articles.  Prolonged 
exposure  to  fresh  air  and  sunlight  will,  however,  aid  in  rendering 
them  harmless;  and  can  be  practised  when  it  is  not  certain  that  they 
are  actually  infective.  Infective  articles  of  wool  may  be  sent  to 
the  city  hospital  and  baked. 

The  doctor  visiting  a  case  of  dangerous  infection  or  a  hospital 
for  infectious  diseases  should  cover  his  clothing  with  a  linen  or 
cotton  gown,  and  protect  his  hair  with  a  cap,  these  articles  being 
disinfected  after  the  visit.  By  such  precautions  he  will  avoid  spread- 
ing infection  among  his  patients  or  carrying  it  to  his  own  family. 

The  Furniture,  etc. — The  destruction  of  infective  furniture  is 
unnecessary.  The  doctor  treating  a  case  of  infectious  disease,  if 
he  properly  perform  his  functions,  will  save  much  trouble  and  money 


Disinfection  of  the  Patient  185 

for  his  patient  by  ordering  his  immediate  isolation  in  an  uncarpeted, 
scantily  and  simply  furnished  room  the  moment  an  infectious  dis- 
ease is  suspected.  However,  if  before  his  removal  the  patient  has 
occupied  another  bed,  its  clothing  should  be  promptly  disinfected. 

After  the  recovery  or  death  of  the  patient  the  walls  and  ceiling 
of  the  room  should  be  sprayed  with  a  formaldehyd  solution,  or  the 
room  sealed  and  filled  with  the  vapor.  If  they  are  hung  with 
paper,  they  should  be  dampened  with  1:1000  bichlorid  of  mercury 
solution  before  new  paper  is  hung. 

Strehl  has  demonstrated  that  when  10  per  cent,  formalin  solution 
is  sponged  upon  artificially  infected  curtains,  etc.,  the  bacteria 
are  killed.  This  is  an  important  adjunct  to  our  means  of  disinfect- 
ing the  furniture  of  the  sick-chamber. 

The  floor  should  be  scoured  with  40  per  cent,  formaldehyd  solu- 
tion, 5  per  cent,  carbolic  acid  solution,  or  i :  1000  bichlorid  of  mercury 
solution  (no  soap  being  used,  as  it  destroys  the  bichlorid  of  mercury 
and  prevents  its  action),  and  all  the  wooden  articles  wiped  off  two 
or  three  times  with  one  of  the  same  solutions.  If  a  straw  mattress 
was  used  it  should  be  burned  and  the  cover  boiled.  If  a  hair  mat- 
tress was  used,  it  can  be  steamed  or  baked  by  the  manufacturers, 
who  usually  have  ovens  for  the  purpose  of  destroying  moths,  but 
which  answer  for  sterilizing  closets.  Curtains,  shades,  etc.,  should 
receive  proper  attention;  but,  of  course,  the  greater  the  precautions 
exercised  in  the  beginning,  the  fewer  the  articles  that  will  need  at- 
tention in  the  end. 

The  Patient,  whether  he  live  or  die,  may  be  a  means  of  spreading 
the  disease  unless  specially  cared  for.  After  convalescence  the 
body  should  be  scoured  with  biniodide  of  mercury  soap,  bathed 
with  a  weak  bichlorid  of  mercury  solution  or  with  a  2  per  cent,  car- 
bolic acid  solution,  or  with  25-50  per  cent,  alcohol,  before  the  pa- 
tient is  allowed  to  mingle  with  society,  and  the  hair  should  either 
be  cut  off  or  carefully  washed  with  the  disinfecting  solution  or  an 
antiseptic  soap.  In  desquamative  diseases  it  seems  best  to  have 
the  entire  body  anointed  with  cosmolin  once  daily,  beginning 
before  desquamation  begins  and  having  the  unguent  well  rubbed  in, 
in  order  to  prevent  the  particles  of  epidermis,  in  which  the  specific 
contagium  probably  occurs,  being  distributed  through  the  atmos- 
phere. Carbolated  may  be  better  than  plain  cosmolin,  not  be- 
cause of  the  very  slight  antiseptic  value  it  possesses,  but  because  it 
helps  to  allay  the  itching  which  may  accompany  the  desquamative 
process. 

.After  the  patient  is  about  again,  common  sense  will  prohibit 
the  admission  of  visitors  until  the  suggested  disinfective  measures 
have  been  adopted,  and  after  this,  touching,  and  especially 'kissing 
him,  should  be  avoided  for  some  time. 

The  bodies  of  those  that  die  of  infectious  diseases  should  be  washed 
in  a  strong  disinfectant  solution,  and  given  a  strictly  private  funeral. 


1 86  Sterilization  and  Disinfection 

If  this  be  impossible,  the  body  should  be  embalmed,  sealed  in  the 
coffin,  and  the  face  viewed  through  a  plate  of  glass;  the  body  is 
best  disposed  of  by  cremation,  though  it  is  not  rarely  necessary  as 
a  dead  body  cannot  remain  a  source  of  infection  for  an  indefinite 
period.  Esmarch,*  who  made  a  series  of  laboratory  experiments 
to  determine  the  fate  of  pathogenic  bacteria  in  the  dead  body, 
found  that  in  septicemia,  cholera,  anthrax,  malignant  edema,  tuber- 
culosis, tetanus,  and  typhoid  fever  the  pathogenic  bacteria  all  die 
sooner  or  later,  more  rapidly  during  active  decomposition  than  during 
preservation  of  the  tissues. 

*  "Zeitschrift  fur  Hygiene,"  1893. 


CHAPTER  VII 

CULTURE-MEDIA    AND    THE    CULTIVATION    OF    MICRO- 
ORGANISMS 

IN  order  to  observe  them  accurately  micro-organisms  must  be 
separated  from  their  natural  surroundings  and  artificially  cultivated 
upon  certain  prepared  media  of  standard  composition.  The  effects 
of  one  organism  upon  the  growth  of  another,  by  neutralizing  its 
metabolic  products,  by  changing  the  reaction  of  the  medium  in 
which  it  grows  so  as  to  inhibit  further  multiplication,  by  dissolving 
the  other  species  through  its  enzymes,  etc.,  suffice  to  show  how 
impossible  it  is  to  determine  the  natural  history  of  any  organism 
unless  it  be  kept  strictly  away  from  other  species. 

Fortunately  the  same  general  principles  apply  equally  for  the 
cultivation  of  all  forms  of  micro-organismal  life,  and  much  the  same 
media  apply  in  all  cases.  What  is  said,  therefore,  about  the  bacteria 
may  be  regarded  as  appropriate  for  all. 

BACTERIA 

Various  organic  and  inorganic  mixtures  have  been  suggested  for 
the  cultivation  of  bacteria,  but  few  have  met  with  particular  favor 
and  become  standards.  At  the  present  time  certain  standard  media 
are  used  in  every  laboratory  in  the  world;  all  systematic  study  of 
the  organisms  depends  upon  the  behavior  of  bacteria  upon  them, 
and  no  study  of  micro-organisms  can  be  regarded  as  complete  unless 
the  behavior  of  the  bacteria  upon  them  has  been  carefully  considered. 

Our  studies  of  the  biology  of  the  bacteria  have  shown  that  they 
grow  best  in  mixtures  containing  at  least  80  per  cent,  of  water,  of 
neutral  or  feebly  alkaline  reaction,  and  of  a  composition  which,  for 
the  pathogenic  forms  at  least,  should  approximate  the  juices  of  the 
animal  body.  It  might  be  added  that  transparency  is  a  very  desir- 
able quality,  and  that  the  most  generally  useful  culture-media  are 
those  that  can  be  liquefied  and  solidified  at  will. 

All  accurate  bacteriologic  culture  experiments  require  that  an 
exact  knowledge  of  the  chemistry  of  the  media  used  shall  be  at  hand. 
The  importance  of  this  knowledge  is  suggested  by  the  pains  taken 
to  arrive  at  it.  The  best  bacteriologists  of  America  have  agreed 
upon  certain  details  that  are  explained  in  the  following  excerpts 
from  the  Report  of  the  Committee  of  Bacteriologists  of  the  American 
Public  Health  Association.* 

*  "Jour.  Amer.  Public  Health  Assoc.,"  Jan.,  1898,  p.  72. 
187 


1 88 


Cultivation  of  Micro-organisms 


"The  first  thing  to  obtain  is  a  standard  'indicator'  which  will  give  uniform 
results.  These  requirements  are  best  fulfilled  by  phenolphthalein." 

"The  question  of  the  proper  reaction  of  media. for  the  cultivation  of  bacteria 
and  the  method  of  obtaining  this  reaction  have'  been  discussed  in  a  valuable 
paper  by  Mr.  George  W.  Fuller,  published  in  the  'Journal  of  the  American  Public 
Health  Association,'  Oct.,  1895,  vol.  xx,  p.  321." 

"Method  of  determining  the  degree  of  reaction  of  culture-media:  For  this 
most  important  part  in  the  preparation  of  culture-media,  burets  graduated  into 
one-tenth  c.c.  and  three  solutions  are  required — 


Fig.  43. — Buret  for  titrating  media.     (From  Hiss  and  Zinsser,  "Text-Book  of 
Bacteriology,"  D.  Appleton  &  Co.,  Publishers.) 

"i.  A  0.5  per  cent,  solution  of  commercial  phenolphthalein,  in  50  per  cent, 
alcohol. 

"2.  A —  solution  of  sodium  hydroxid. 

"3.  A —  solution  of  hydric  chlorid. 
20 

"Solutions  2  and  3  must  be  accurately  made  and  must  correspond  with  the 
normal  solutions  soon  to  be  referred  to. 

"Solutions  of  sodium  hydroxid  are  prone  to  deterioration  from  the  absorption 
of  carbon  dioxid  and  the  consequent  formation  of  sodium  carbonate.  To  pre- 
vent as  much  as  possible  this  change,  it  is  well  to  place  in  the  bottle  containing 
the  stock  solution  a  small  amount  of  calcium  hydroxid,  while  the  air  entering  the 
burets  or  the  supply  bottles  should  be  made  to  pass  through  a  U-tube  containing 
caustic  soda,  to  extract  from  it  the  carbon  dioxid." 

"The  medium  to  be  tested,  all  ingredients  being  dissolved,  is  brought  to  the 


Cultivation  of  Bacteria  189 

prescribed  volume  by  the  addition  of  distilled  water  to  replace  that  lost  by  boiling, 
and  after  being  thoroughly  stirred,  5  cc.  are  transferred  to  a  6-inch  porcelain 
evaporating-dish.  To  this  45  cc.  of  distilled  water  are  added  and  the  50  cc. 
of  fluid  are  boiled  for  three  minutes  over  a  flame.  One  cubic  centimeter  of  the 
solution  of  phenolphthalein  (No.  i)  is  then  added,  and  by  titration  with  the 
required  reagent  (No.  2  or  No.  3)  the  reaction  is  determined.  In  the  majority  of 

instances  the  reaction  will  be  found  to  be  acid,  so  that  the  —  sodium  hydroxid 

is  the  reagent  most  frequently  required.  This  determination  should  be  made 
not  less  than  three  times  and  the  average  of  the  results  obtained  taken  as  the 
degree  of  the  reaction. 

"One  of  the  most  difficult  things  to  determine  in  this  process  is  exactly  when 
the  neutral  point  is  reached  as  shown  by  the  color  developed,  and  to  be  able  in 
every  instance  to  obtain  the  same  shade  of  color.  To  aid  in  this  regard,  it  may 
be  remarked  that  in  bright  daylight  the  first  change  that  can  be  seen  on  the  addi- 
tion of  alkali  is  a  very  faint  darkening  of  the  fluid,  which,  on  the  addition  of  more 
alkali,  becomes  a  more  evident  color  and  develops  into  what  might  be  described 
as  an  Italian  pink.  A  still  further  addition  of  alkali  suddenly  develops  a  clear 
and  bright  pink  color,  and  this  is  the  reaction  always  to  be  obtained.  All  titra- 
tions  should  be  made  quickly  and  in  the  hot  solutions  to  avoid  complications 
arising  from  the  presence  of  carbon  dioxid. 

"The  next  step  in  the  process  is  to  add  to  the  bulk  of  the  medium  the  calcu- 
lated amount  of  the  reagent,  either  alkali  or  acid,  as  may  be  determined.  For  the 
purpose  of  neutralization  a  normal  solution  of  sodium  hydroxid  or  of  hydric 
chlorid  is  used,  and  after  being  thoroughly  stirred  the  fluid  thus  neutralized  is 
again  tested  in  the  same  manner  as  at  first,  to  insure  the  proper  reaction  of  the 
medium  being  attained.  When  neutralization  is  to  be  effected  by  the  addition  of 
an  alkali,  it  not  infrequently  happens  that  after  the  calculated  amount  of  normal 
solution  of  sodium  hydroxid  has  been  added,  the  second  test  will  show  that  the 
medium  is  acid  to  phenolphthalein,  to  the  extent  sometimes  of  0.5  to  i  per  cent. 
This  discrepancy  is  perhaps  due  to  side  reactions  which  are  not  understood.  The 
reaction  of  the  medium,  however,  must  be  brought  to  the  desired  point  by  the 
further  addition  of  sodium  hydroxid,  and  the  titrations  and  additions  of  alkali 
must  be  repeated  until  the  medium  has  the  desired  reaction  (i.e.,  o.o  per  cent,  to 
0.005  per  cent.;  see  below). 

"After  the  prescribed  period  of  heating,  it  is  frequently  found  that  the  medium 
is  again  slightly  acid,  usually  about  0.5  per  cent.  Without  correcting  this,  the 
fluid  is  to  be  filtered  and  the  calculated  amount  of  acid  or  alkali  is  to  be  added 
to  change  the  reaction  to  the  one  desired.  A  still  further  change  in  reaction  is 
not  infrequently  to  be  observed  after  sterilization,  the  degree  of  acidity  varying 
apparently  with  the  composition  of  the  media  and  the  degree  and  continuance 
of  the  heat." 

"Manner  of  expressing  the  reaction:  Since  at  the  time  the  reaction  is  first 
determined  culture-media  are  more  often  acid  than  alkaline,  it  is  proposed  that 
acid  media  be  designated  by  the  plus  sign  and  alkaline  media  by  the  minus  sign, 
and  that  the  degree  of  acidity  or  alkalinity  be  noted  in  parts  per  hundred.  Thus, 
a  medium  marked  +  1.5  would  indicate  that  the  medium  was  acid,  and  that  1.5 

per  cent,  of  —  sodium  hydroxid  is  required  to  make  it  neutral  to  phenolphthalein; 
while  —  1.5  would  indicate  that  the  medium  was  alkaline  and  that  1.5  per  cent,  of 

-  acid  must  be  added  to  make  it  neutral  to  the  indicator." 

i 

"Standard  reaction  of  media  (provisional}: 

"Experience  seems  to  vary  somewhat  as  to  the  optimum  degree  of  reaction 
which  shall  be  uniformly  adopted  in  the  preparation  of  standard  culture-media. 
To  what  extent  this  is  due  to  variation  in  natural  conditions  as  compared  with 
variations  of  laboratory  procedure  it  seems  impossible  to  state.  Somewhat 
different  degrees  of  reaction  for  optimum  growth  are  required,  not  only  in  or 
upon  the  media  of  different  composition  and  by  bacteria  of  different  species,  but 
also  by  bacteria  of  the  same  species  when  in  different  stages  of  vitality.  The 
bulk  of  available  evidence  from  both  Europe  and  America  points  to  a  reaction 
of  +1.5  as  the  optimum  degree  of  reaction  for  bacterial  development  in  inoculated 
culture  media.  While  this  experience  is  at  variance  with  that  in  several  of  our 
own  laboratories,  it  has  been  deemed  wisest  to  adopt  +1.5  as  the  provisional 


1 90  Cultivation  of  Micro-organisms 

standard  reaction  of  media,  but  with  the  recommendation  that  the  optimum 
growth  reaction  be  always  recorded  with  the  species." 

BOUILLON 

This  is  one  of  the  most  useful  and  most  simple  media.  It  can  be 
prepared  from  meat  or  from  meat  extract,  and  is  the  basis  of  most 
of  the  culture-media.  The  addition  of  10  per  cent,  of  gelatin  makes 
it  "gelatin;"  that  of  i  per  cent,  of  agar-agar  makes  it  "agar-agar." 

I.  To  Prepare  Bouillon  from  Fresh  Meat. — To  500  grams  of  finely 
chopped  lean,  boneless  beef,  1000  cc.  of  clean  water  are  added  and 
allowed  to  stand  for  about  twelve  hours  on  ice.  At  the  end  of  this 
time  the  liquor  is  decanted,  that  remaining  on  the  meat  expressed 
through  a  cloth,  and  then,  as  the  entire  quantity  is  seldom  regained, 
enough  water  added  to  bring  the  total  amount  up  to  1000  cc.  This 
liquid  is  called  the  meat-infusion.  To  it  10  grams  of  Witte's  or 
Fairchild's  dried  beef-peptone  and  5  grams  of  sodium  chlorid  are 
added,  and  the  whole  boiled  until  the  albumins  of  the  meat-infusion 
coagulate,  titrated  or  otherwise  corrected  for  acidity,  boiled  again 
for  a  short  time,  and  then  filtered  through  a  fine  filter  paper.  It 
should  be  slightly  yellow  and  perfectly  clear  and  limpid.  Smith,* 
referring  to  bouillon  intended  for  the  culture  of  diphtheria  bacilli 
for  toxin,  says  that  when  the  peptones  are  added  before  boiling 
most  of  them  are  lost,  and  therefore  recommends  that  the  meat- 
infusion  be  boiled  and  filtered  and  the  solid  ingredients  added  and 
dissolved  subsequently.  The  reaction,  which  is  strongly  acid,  is 
then  carefully  corrected  by  titration  according  to  the  directions 
already  given. 

For  rough  work  in  students'  classes  litmus  paper  may  be  used  as 
an  indicator  for  determining  and  correcting  the  acidity  resulting 
from  the  sarcolactic  and  other  acids  in  the  meat-infusion,  the  alka- 
line solution  being  added  drop  by  drop  until  a  faint  blue  appears  on 
the  red  paper;  or  phenolphthalein  can  be  employed,  the  addition  of 
the  alkaline  solution  being  continued  until  a  drop  of  the  bouillon 
produces  a  red  spot  upon  phenolphthalein  paper,  made,  as  suggested 
by  Timpe,  by  saturating  bibulous  paper  cut  into  strips  with  a  solu- 
tion of  5  grams  of  phenolphthalein  to  i  liter  of  50  per  cent,  alcohol. 
Acids  do  not  change  the  appearance  of  the  paper,  but  small  traces 
of  alkali  turn  it  red. 

If  the  bouillon  is  to  be  employed  for  exact  work,  these  crude 
methods  should  not  be  adopted,  but  chemical  titration  according  to 
the  method  already  given  should  be  performed.  After  titration  the 
bouillon  must  again  be  boiled  for  a  few  minutes,  in  order  to  precipi- 
tate the  acid  albumins,  as  much  water  added  as  has  been  lost  by 
evaporation,  and  the  fluid  filtered  through  a  pharmaceutic  filter. 

The  filtered  fluid  is  dispensed  in  previously  sterilized  tubes  with 
cotton  plugs — about  10  cc.  to  each — or  in  flasks,  and  is  then  sterilized 
*  " Trans.  Assoc.  Amer.  Phys.,"  1896. 


To  Prepare  Bouillon  from  Meat  Extract 


191 


by  steam  three  successive  days  for  fifteen  to  twenty  minutes  each, 
according  to  the  directions  already  given  for  intermittent  steriliza- 
tion, or  superheated  in  the  autoclave. 

The  loss  of  water  during  boiling  is  an  important  matter  to  bear 
in  mind,  as  unless  properly  replaced  it  is  the  cause  of  disproportion 
between  the  fluids  and  solids  of  the  media.  The  quantity  must 
therefore  be  measured  before  filtration  and  enough  water  added  to 
replace  what  has  been  lost.  Measuring  before  filtration  is  compara- 
tively easy  with  bouillon,  but  difficult  with  heavy  liquids,  like  the 
gelatin  and  agar-agar  solutions.  To  overcome  this  difficulty  it  is 


Fig.  44. — Funnel  for  filling  tubes  with  culture  media  (Warren):  a,  Funnel 
containing  the  culture  media  in  liquid  condition;  b,  pinch-cock  by  which  the  flow 
of  fluid  into  the  test-tube  is  regulated;  c,  rubber  tubing. 

best  to  make  the  entire  preparation  by  weight  and  not  by  volume. 
A  pair  of  platform  scales  with  sliding  indicators  will  first  balance 
the  empty  kettle  and  then  show  the  correct  quantity  of  each  added 
ingredient.  After  boiling,  the  kettle  can  be  returned  to  the  scale 
and  the  exact  quantity  of  water  to  be  added  determined. 

II.  To  Prepare  Bouillon  from  Meat  Extract. — When  desirable,  the 
bouillon  may  also  be  prepared  from  beef-extract,  the  method  being 
very  simple:  To  1000  cc.  of  clean  water  10  grams  of  Witte's  dried 
beef-peptone,  5  grams  of  sodium  chlorid,  and  about  2  grams  of  beef- 


192  Cultivation  of  Micro-organisms 

extract  are  added.  The  solution  is  boiled  until  the  constituents 
are  dissolved,  titrated,  and  filtered  when  cold.  If  it  be  filtered  while 
hot,  there  is  always  a  subsequent  precipitation  of  meat-salts,  which 
clouds  it. 

Bouillon  and  other  liquid  culture  media  are  best  dispensed  and 
kept  in  small  receptacles — test-tubes  or  flasks — in  order  that  a  single 
contaminating  organism,  should  it  enter,  may  not  spoil  the  entire 
quantity.  A  convenient,  simple  apparatus  for  filling  tubes  with 
liquid  media  consists  of  a  funnel  to  which  a  short  glass  pipet  is  at- 
tached by  a  bit  of  rubber  tubing.  A  pinch-cock  controls  the  outflow 
of  the  liquid.  The  apparatus  need  not  be  sterilized  before  using,  as 
the  culture  medium  must  subsequently  be  sterilized  either  by  the 
intermittent  method  or  in  the  autoclave  after  the  tubes  are  filled. 
The  test-tubes  and  flasks  into  which  the  culture  medium  is  filled 
must,  however,  be  previously  sterilized  by  dry  heat,  unless  the  sub- 
sequent sterilization  is  to  be  performed  in  the  autoclave,  when  it 
may  be  unnecessary. 

Sugar  bouillon  is  bouillon  containing  in  solution  known  percent- 
ages of  such  sugars  as  glucose,  lactose,  saccharose,  etc.  As  Smith* 
has  pointed  out,  if  the  quantity  of  sugar  in  the  bouillon  is  to  be  ac- 
curately known,  it  is  necessary  to  first  destroy  the  muscle  sugars  in 
the  meat-infusion.  This  can  be  done  by  adding  a  culture  of  the 
colon  bacillus  to  the  meat-infusion  and  permitting  fermentation  to 
continue  overnight,  then  finishing  the  bouillon  and  adding  the 
known  quantity  of  whatever  sugar  is  desired.  About  i  per  cent, 
of  dextrose,  lactose,  saccharose  or  galactose  is  all  that  is  required. 
More  may  be  injurious.  If  the  bouillon  be  made  from  meat  ex- 
tract, fermentation  may  not  be  necessary. 

The  sugar  bouillons  should  not  be  sterilized  in  the  autoclave,  as 
the  high  temperatures  chemically  alter  the  sugars. 

GELATIN 

The  culture-medium  known  as  gelatin  is  bouillon  to  which  10 
per  cent,  of  gelatin  is  added.  It  has  the  decided  advantage  over 
bouillon  that  it  is  not  only  an  excellent  food  for  bacteria,  and,  like 
the  bouillon,  transparent,  but  also  is  solid  at  the  room  temperature. 
Nor  is  this  all:  it  is  a  transparent  solid  that  can  be  made  liquid  or 
solid  at  will.  Leffmann  and  La  Wall  have  examined  commercial 
gelatins  and  found  that  many  of  them  contain  sulphur  dioxid  in 
quantities  as  great  as  835  parts  per  million.  As  the  varying  quan- 
tity of  this  impurity  may  modify  the  growth  of  the  culture,  pure 
gelatin  should  be  demanded,  and  all  gelatin  should  be  washed  for 
some  hours  in  cold  running  water  after  being  weighed  and  before 
being  added  to  the  bouillon.  It  is  prepared  as  follows: 

To  1 600  cc.  of  meat-infusion  or  to  1000  cc.  of  water  containing 
*  "Jour,  of  Exp.  Med.,"  n,  No.  5,  p.  546. 


Agar-agar  193 

2  grams  of  beef-extract  in  solution,  10  grams  of  peptone,  5  grams  of 
salt,  and  100  grams  of  gelatin  ("Gold  label"  is  the  best  commercial 
article)  are  added,  and  heated  until  the  ingredients  are  dissolved. 
The  solution  reacts  strongly  acid  and  must  be  corrected  by  titra- 
tion,  as  already  described.  It  must  then  be  returned  to  the  fire 
and  boiled  for  about  an  hour.  As  gelatin  is  apt  to  burn  when  boiled 
over  the  direct  flame,  double  boilers  have  been  suggested,  but  unless 
the  outer  kettle  is  filled  with  brine  or  saturated  calcium  chlorid  solu- 
tion, they  are  very  slow,  and  when  proper  care  is  exercised  there  is 
really  no  great  danger  of  the  gelatin  burning.  It  must  be  stirred 
occasionally,  and  the  flame  should  be  so  distributed  by  wire  gauze 
or  by  placing  a  sheet  of  asbestos  between  it  and  the  kettle  as  not  to 
act  upon  a  single  point.  At  the  end  of  the  hour  the  albumins 
of  the  meat-infusion  will  be  coagulated  and  the  gelatin  thoroughly 
dissolved.  Giinther  has  shown  that  the  gelatin  congeals  better 
if  allowed  to  dissolve  slowly  in  warm  water  before  boiling.  As 
much  water  as  has  been  lost  by  vaporization  during  the  process  of 
boiling  should  be  replaced.  It  is  well  to  cool  the  liquid  to  about 
6o°C.,  add  the  water  mixed  with  the  white  of  an  egg  to  clear  the 
liquid,  boil  again  for  half  an  hour,  and  filter. 

If  the  filter  paper  be  of  good  quality,  properly  folded  (pharma- 
ceutic  filter),  wet  with  boiling  water,  and  if  the  gelatin  be  properly 
dissolved,  the  whole  quantity  should  pass  through  before  cooling 
too  much.  Should  only  half  go  through  before  cooling,  the  re- 
mainder must  be  returned  to  the  pot,  heated  to  boiling  once  more, 
and  then  passed  through  a  new  filter  paper.  As  a  matter  of  fact, 
gelatin  usually  filters  readily.  A  wise  precaution  is  to  catch  the 
first  few  centimeters  in  a  test-tube  and  boil  them,  so  that  if  cloudi- 
ness show  the  presence  of  uncoagulated  albumin,  the  whole  mass 
can  be  boiled  again.  The  finished  gelatin,  which  is  perfectly  trans- 
parent and  of  an  amber  color,  is  at  once  distributed  into  sterilized 
tubes  and  sterilized  like  the  bouillon  by  the  intermittent  method. 
The  sterilization  can  also  be  satisfactorily  performed  by  the  use  of 
the  autoclave  at  iio°-ii5°C.  for  fifteen  minutes,  but  this  method  is 
probably  less  well  adapted  to  the  sterilization  of  gelatin  than  of  the 
other  media,  as  the  high  degree  of  heat  injures  its  subsequent  solidi- 
fying power. 

Gelatin  becomes  liquid  at  37°C.  It  cannot,  therefore,  be  used 
with  advantage  for  cultures  that  must  be  kept  at  body  temperatures. 

AGAR-AGAR 

Agar-agar  is  the  commercial  name  of  a  preparation  made  from  a 
Ceylonese  sea-weed.  It  reaches  the  market  in  the  form  of  long 
shreds  of  semi-transparent,  isinglass-like  material,  less  commonly  in 
long  bars  of  compressed  flakes,  rarely  in  the  form  of  powder.  It 
dissolves  slowly  in  boiling  water  with  a  resulting  thick  jelly  when 
13 


194  Cultivation  of  Micro-organisms 

cold.  The  jelly,  which  solidifies  between  40°  and  5o°C.,  cannot 
again  be  melted  except  by  the  elevation  of  its  temperature  to  the 
boiling-point.  The  culture-medium  made  from  agar-agar  is  nearly 
transparent.  In  addition  to  its  ability  to  liquefy  and  solidify,  it 
has  the  advantage  of  remaining  solid  at  comparatively  high  tem- 
peratures so  as  to  permit  keeping  the  cultures  grown  upon  it  at  the 
incubation  temperature, — i.e.,  37°C., — at  which  temperature  gelatin 
is  always  liquid. 

The  preparation  of  agar-agar  is  commonly  described  in  the  text- 
books as  one  "requiring  considerable  patience  and  much  waste  of 
filter  paper."  In  reality,  it  is  not  difficult  if  a  good  heavy  filter  paper 
be  obtained  and  no  attempt  made  to  filter  the  solution  until  the  agar- 
agar  is  perfectly  dissolved. 

It  is  prepared  as  follows:  To  1000  cc.  of  bouillon  made  as  described 
above,  preferably  of  meat  instead  of  beef-extract,  10  to  15  grams  of 
agar-agar  are  added.  The  mixture  is  boiled  vigorously  for  an  hour 
in  an  open  pot  over  the  direct  gas  flame  or  in  the  double  boiler  with 
saturated  calcium  chlorid  solution  in  the  outside  pot.  After  being 
cooled  to  about  6o°C.,  and  after  the  correction  of  the  reaction  by 
titration,  an  egg  beaten  up  in  water  is  added,  and  the  liquid  again 
boiled  until  the  egg-albumen  is  entirely  coagulated. 

After  the  second  boiling  and  the  replacement  of  the  volatilized 
water,  the  agar-agar  is  filtered  through  a  carefully  folded  pharma- 
ceutic  filter  wet  with  boiling  water.  It  may  expedite  matters  to 
pour  in  about  one-half  of  the  solution,  keep  the  remainder  hot,  and 
subsequently  add  it. 

The  formerly  much  employed  hot-water  and  gas-jet  filters  are  un- 
necessary. If  properly  prepared,  the  whole  quantity  will  filter  in 
from  fifteen  to  thirty  minutes. 

Ravenel*  prepares  agar-agar  by  making  two  solutions,  one  repre- 
senting the  meat-infusion,  but  twice  the  usual  strength,  the  other 
the  agar-agar  dissolved  in  one-half  the  usual  quantity  of  water* 
The  agar-agar  is  dissolved  by  exposure  to  superheated  steam  in  the 
autoclave,  after  which  the  two  solutions  are  poured  together  and 
boiled  until  all  of  the  albumins  are  precipitated.  The  coagulation 
of  the  albumins  of  the  meat-infusion  serves  to  clarify  the  agar-agar. 

If  agar-agar  is  to  be  made  with  beef-extract,  the  bouillon  should 
be  made  first  and  filtered  when  cold,  to  exclude  the  uratic  salts  which 
otherwise  precipitate  in  the  agar-agar  when  cold  and  form  an  un- 
sightly cloud. 

The  finished  agar-agar  should  be  a  colorless,  nearly  transparent, 
firm  jelly.  It  is  dispensed  in  tubes  like  the  gelatin  and  bouillon, 
sterilized  by  steam,  either  by  the  intermittent  process  or  in  the  auto- 
clave, and  after  the  last  sterilization,  before  cooling,  each  tube  is 
inclined  against  a  slight  elevation,  so  as  to  permit  the  jelly  to  solidify 
obliquely  and  afford  an  extensive  flat  surface  for  the  culture. 
*  "Journal  of  Applied  Microscopy,"  June,  1898,  vol.  I,  No.  6,  p.  106. 


Blood-serum  195 

After  the  agar-agar  jelly  solidifies  it  retracts  so  that  a  little  water 
collects  at  the  lower  part  of  the  tube.  This  should  not  be  removed, 
as  it  keeps  the  jelly  moist,  and  also  distinctly  influences  the  character 
of  the  growth  of  the  bacteria. 

Glycerin  Agar-agar.— Certain  bacteria  among  which  is  the 
tubercle  bacillus,  will  not  grow  upon  agar-agar  prepared  as  described 
above,  but  will  do  so  if  3  to  7  per  cent,  of  glycerin  be  added  after 
filtration.  This  fact  was  discovered  by  Roux  and  Nocard. 

Blood  Agar-agar  was  recommended  by  R.  Pfeiffer  for  the  culti- 
vation of  the  influenza  bacillus.  It  is  ordinary  agar-agar  whose 
surface  is  coated  with  a  little  blood  secured  under  aseptic  precautions 
from  the  finger-tip,  ear-lobule,  etc.,  of  man,  or  from  the  vein  of  one 
of  the  lower  animals.  Some  bacteriologists  prepare  a  hemoglobin 
agar-agar  by  spreading  a  little  powdered  hemoglobin  upon  the  surface 
of  the  agar-agar.  As  powdered  hemoglobin  is  not  sterile,  the  medium 
must  be  sterilized  after  its  addition. 

The  blood  agar-agar  should  be  kept  in  the  incubator  a  day  or  two 
before  use  so  as  to  insure  perfect  sterility. 

BLOOD-SERUM 

The  advantage  possessed  by  this  medium  is  that  it  is  primarily  a 
constituent  of  the  animal  body,  and  hence  offers  conditions  favor- 
able for  the  development  of  the  parasitic  forms  of  bacteria.  If  the 
blood-serum  is  to  be  employed  fresh,  it  must  either  be  heated  or  kept 
sufficiently  long  to  lose  its  natural  germicidal  properties.  The 
statement  that  serum  represents  the  normal  body- juice  is  erroneous, 
as  it  is  minus  the  fibrin  factors  and  some  of  the  salts,  and  contains 
new  bodies  liberated  from  the  destroyed  leukocytes.  Solidified 
blood-serum,  exposed  to  the  heat  of  the  sterilizing  apparatus,  in  no 
sense  resembles  the  body- juices. 

It  is  one  of  the  most  difficult  media  to  prepare.  The  blood  must 
be  obtained  either  by  bleeding  some  good-sized  animal,  or  from  a 
slaughter-house,  in  appropriate  receptacles,  the  best  things  for  the 
purpose  being  i -quart  fruit  jars  with  tightly  fitting  lids.  The  jars 
are  sterilized  by  heat,  closed,  and  carried  to  the  slaughter-house, 
where  the  blood  is  permitted  to  flow  into  them  from  the  severed 
vessels  of  the  animal.  It  seems  advisable  to  allow  the  first  blood  to 
escape,  as  it  is  likely  to  become  contaminated  from  the  hair.  By 
waiting  until  a  coagulum  forms  upon  the  hair  the  danger  of  con- 
tamination is  diminished.  The  jars,  when  full,  are  allowed  to  stand 
undisturbed  until  firm  coagula  form  within  them,  after  which  they 
are  carried  to  the  laboratory  and  stood  upon  ice  for  forty-eight 
hours,  by  which  time  the  clots  will  have  retracted  considerably,  and 
a  moderate  amount  of  clear  serum  can  be  removed  by  sterile  pipets 
and  placed  in  sterile  tubes.  If  the  serum  obtained  be  red  and 
clouded  from  the  presence  of  corpuscles,  it  may  be  pipetted  into 


196  Cultivation  of  Micro-organisms 

sterile  cylinders  and  allowed  to  sediment  for  twelve  hours,  then 
repipetted  into  tubes. 

As  the  demand  for  serum  has  been  considerable  during  the  last 
few  years,  commercial  houses  dealing  in  biologic  products  now 
market  fresh  horse  serum,  preserved  with  chloroform,  in  liter  bottles. 
This  can  be  employed  with  great  satisfaction,  the  chloroform  being 
driven  off  during  coagulation  and  sterilization. 

If  it  be  desirable  to  use  the  serum  as  a  liquid  medium,  it  is  exposed 
to  a  temperature  of  6o°C.  for  one  hour  upon  each  of  five  consecutive 
days.  To  coagulate  the  serum  and  make  a  solid  culture  medium, 
it  may  be  exposed  twice,  for  an  hour  each  time — or  three  times  if 
there  be  reason  to  think  it  badly  contaminated — to  a  temperature 


Fig.  45. — Koch's  apparatus  for  coagulating  and  sterilizing  blood-serum. 

just  short  of  the  boiling-point.  During  the  process  coagulation 
occurs,  and  the  tubes  should  be  inclined,  so  as  to  offer  an  oblique 
surface  for  the  growth  of  the  organisms.  The  serum  thus  prepared 
should  be  white,  but  may  have  a  reddish-gray  color  if  many  red 
corpuscles  be  present.  It  is  always  opaque  and  cannot  be  melted; 
once  solid,  it  remains  so. 

Koch  devised  a  special  apparatus  for  coagulating  blood-serum. 
The  bottom  should  be  covered  with  wet  cotton,  a  single  layer  of 
tubes  placed  upon  it,  the  glass  lid  closed  and  covered  with  a  layer 
of  felt,  and  the  temperature  elevated  until  coagulation  occurs.  The 
repeated  sterilizations  may  be  conducted  in  this  same  apparatus, 
or  may  be  done  equally  well  in  a  steam  apparatus,  the  cover  of  which 
is  not  completely  closed,  for  if  the  temperature  of  the  serum  be 
raised  too  rapidly  it  is  certain  to  bubble,  so  that  the  desirable  smooth 
surface,  upon  which  the  culture  is  to  be  made,  is  ruined. 

Like  other  culture-media,  blood-serum  and  its  combinations  may 
be  sterilized  in  the  autoclave  and  much  time  thus  saved.  The  serum 
should,  however,  first  be  coagulated,  else  bubbling  is  apt  to  occur 


Potatoes  197 

and  ruin  its  surface.  The  autoclave  temperature  unfortunately 
makes  the  preparation  very  firm  and  hard,  considerable  fluid  being 
pressed  out  of  it. 

It  is  said  that  considerable  advantage  is  secured  from  the  addition 
of  neutrose  to  blood-serum,  which  prevents  its  coagulating  when 
heated.  It  can  then  be  sterilized  like  bouillon  and  can  subsequently 
be  solidified,  when  desired,  by  the  addition  of  some  agar-agar. 

Fresh  blood-serum  can  be  kept  on  hand  in  the  laboratory,  in 
sterile  bottles,  by  adding  an  excess  of  chloroform.  In  the  process 
of  coagulation  and  sterilization  the  chloroform  is  evaporated;  the 
serum  is  unchanged  by  its  presence. 

Loffler's  Blood-serum  Mixture,  which  seems  rather  better  for  the 
cultivation  of  some  species  than  the  blood-serum  itself,  consists  of 
i  part  of  a  beef-infusion  bouillon  containing  i  per  cent,  of  glucose 
and  3  parts  of  liquid  blood-serum.  After  being  well  mixed  the  fluid 
is  distributed  in  tubes,  and  sterilized  and  coagulated  like  the  blood- 
serum  itself.  As  prepared  by  Lofner  it  was  soft,  semi-gelatinous 
and  semi-transparent,  not  firm  and  white;  therefore  should  be  steril- 
ized at  low  temperatures.  Many  organisms  grow  more  luxuriantly 
upon  it  than  upon  either  plain  blood-serum  or  other  culture  media. 
Its  especial  usefulness  is  for  the  cultivation  of  Bacillus  diphtheriae, 
which  grows  rapidly  and  with  a  characteristic  appearance. 

Alkaline  Blood-serum. — According  to  Lorrain  Smith,  a  very  useful  culture 
medium  can  be  prepared  as  follows:  To  each  100  cc.  of  blood-serum  add  1-1.5 
cc.  of  a  10  per  cent,  solution  of  sodium  hydrate  and  shake  it  gently.  Put  suffi- 
cient of  the  mixture  into  each  of  a  series  of  test-tubes,  and,  laying  them  upon  their 
sides,  sterilize  like  blood-serum,  taking  care  that  their  contents  are  not  heated 
too  quickly,  as  then  bubbles  are  apt  to  form.  The  result  should  be  a  clear,  solid 
medium  consisting  chiefly  of  alkali-albumins.  It  is  especially  useful  for  Bacillus 
diphtheriae. 

Deycke's  Alkali-albuminate. — One  thousand  grams  of  meat  are  macerated  for 
twenty-four  hours  with  1200  cc.  of  a  3  per  cent,  solution  of  potassium  hydrate. 
The  dear  brown  fluid  is  filtered  off  and  pure  hydrochloric  acid  carefully  added 
while  a  precipitate  forms.  The  precipitated  albuminate  is  collected  upon  a  cloth 
filter,  mixed  with  a  small  quantity  of  liquid,  and  made  distinctly  alkaline.  To 
make  solutions  of  definite  strength  it  can  be  dried,  pulverized,  and  redissolved. 

The  most  useful  formula  used  by  Deycke  was  a  2.5  per  cent,  solution  of  the 
alkali-albuminate  with  the  addition  of  i  per  cent,  of  peptone,  i  per  cent,  of 
NaCl,  and  gelatin  or  agar-agar  enough  to  make  it  solid. 

Potatoes. — Without  taking  time  to  review  the  old  method  of 
boiling  potatoes,  opening  them  with  sterile  knives,  and  protecting 
them  in  the  moist  chamber,  or  the  much  more  easily  conducted 
method  of  Esmarch  in  which  the  slices  of  potato  are  sterilized  in  the 
small  dishes  in  which  they  are  afterward  kept  and  used,  we  will  at 
once  pass  to  what  seems  the  most  simple  and  satisfactory  method — 
that  of  Bolton  and  Globig.* 

With  the  aid  of  a  cork-borer  or  Ravenel  potato  cutter  a  little 
smaller  in  diameter  than  the  test-tube  ordinarily  used,  a  number 
of  cylinders  are  cut  from  potatoes.  Rather  large  potatoes  should 

*  "The  Medical  News,"  1887,  vol.  L,  p.  138. 


198  Cultivation  of  Micro-organisms 

be  used,  the  cylinders  being  cut  transversely,  so  that  a  number, 
each  about  an  inch  and  a  half  in  length,  can  be  cut  from  one  potato. 
The  skin  is  removed  from  the  cylinders  by  cutting  off  the  ends,  after 
which  each  cylinder  is  cut  in  two  by  an  oblique  incision,  so  as  to  leave 
a  broad,  flat  surface.  The  half-cylinders  are  placed  each  in  a  test- 
tube  previously  sterilized,  and  are  exposed  three  times,  for  half  an 
hour  each,  to  the  streaming  steam  of  the  sterilizer.  This  steaming 
cooks  the  potato  and  also  sterilizes  it.  Such  potato  cylinders  are 
apt  to  deteriorate  rapidly,  first  by  turning  very  dark,  second  by 
drying  so  as  to  be  useless.  Abbott  has  shown  that  if  the  cut  cylinders 
be  allowed  to  stand  for  twelve  hours  in  running  water  before  being 
dispensed  in  the  tubes,  they  are  not  so  apt  to  turn  dark.  Drying 
may  also  be  prevented  by  adding  a  few  drops  of  clean  water  to  each 
tube  before  sterilizing.  Some  workers  insert  a  bit  of  glass  or  a 
pledget  of  glass  wool  into  the  bottom  of  the  tube  so  as  to  support 
the  potato  and  keep  it  up  out  of  the  water.  It  is  not  necessary  to 
have  a  special  small  chamber  blown  in  the  tube  to  contain  this  water, 

only  a  small  quantity  of  which  need  be  added. 

The  special  reservoir  increases  the  trouble  of 

cleaning  the  tubes. 

If  the  work  to  be  done  with  potatoes  is  to  be 

accurate,  it  is  necessary  to  correct  their  variable 

reaction,  especially  if  the  acids  have  not  been 

sufficiently  removed  by  the  washing  in  running 

water  already  described. 

To  do  this  the  cut  cylinders  are  placed  in  a 

measured    quantity    of    distilled    water    and 
46.— Ravenel's     steamed  for  about  an  hour.     The  reaction  of 
potato  cutter.  the  water  is  then  determined  by  titration  and 

the  desired  amount  of  sodium  hydroxid  added 

to  correct  the  reaction,  after  which  the  potatoes  are  steamed  in  the 
corrected  solution  for  about  thirty  minutes  before  being  placed  in 
the  tubes. 

A  potato-juice  has  also  been  suggested,  and  is  of  some  value. 
It  is  made  thus:  To  300  cc.  of  water  100  grams  of  grated  potato  are 
added,  and  allowed  to  stand  on  ice  over  night.  Of  the  pulp,  300 
cc.  are  expressed  through  a  cloth  and  cooked  for  an  hour  on  a  water- 
bath.  After  cooking,  the  liquid  is  filtered,  titrated  if  desired,  and 
receives  an  addition  of  4  per  cent,  of  glycerin.  Upon  this  medium 
the  tubercle  bacillus  grows  well,  especially  when  the  reaction  of  the 
medium  be  acid. 

Milk. — Milk  is  a  useful  culture-medium.  As  the  cream  which 
rises  to  the  top  is  a  source  of  inconvenience,  it  is  best  to  secure  fresh 
milk  from  which  the  cream  has  been  removed  by  a  centrifugal  ma- 
chine. It  is  given  the  desired  degree  of  alkalinity  by  titration,  dis- 
pensed in  sterile  tubes,  and  sterilized  by  steam  by  the  intermittent 
method  or  in  the  autoclave.  The  opaque  nature  of  this  culture-me- 


Peptone  Solution  199 

dium  often  permits  the  undetected  development  of  contaminating 
organisms.  A  careful  watch  should  therefore  be  kept  lest  it  spoil. 

Litmus  Milk. — This  is  milk  to  which  just  enough  of  a  saturated 
watery  solution  of  pulverized  litmus  is  added  to  give  a  distinct  blue 
color  after  titration.  Litmus  milk  is  probably  the  best  reagent  for 
determining  acid  and  alkali  production  by  bacteria. 

The  watery  solution  of  litmus,  being  a  vegetable  infusion,  is  likely 
to  be  spoiled  by  micro-organismal  growth,  hence  must  be  treated 
like  the  culture  media  and  sterilized  by  steam  every  time  the  recep- 
tacle in  which  it  is  kept  is  opened. 

An  excellent  method  of  preparing  litmus  is  given  by  Prescott  and 
Winslow*  and  is  as  follows: 

To  one-half  pound  of  litmus  cubes  add  enough  water  to  more  than  cover,  boii, 
decant  off  the  solution.  Repeat  this  operation  with  successive  small  quantities 
of  water  until  3  to  4  liters  of  water  have  been  used  and  the  cubes  are  well  ex- 
hausted of  coloring  matter.  Pour  the  decantations  together  and  allow  them  to 
settle  over  night.  Siphon  off  the  clear  solution.  Concentrate  to  about  i  liter  and 
make  the  solution  decidedly  acid  with  glacial  acetic  acid.  Boil  down  to  about 
3-^  liter  and  make  exactly  neutral  with  caustic  soda  or  potash.  To  test  for  the 

neutral  point,  place  one  drop  of  the  solution  in  a  test-tube,  while  one  drop  of  — 
HC1  should  turn  it  red,  one  drop  of  —  NaOHO  should  turn  it  blue.  Filter  the 

20 

solution  and  sterilize  at  no°C.  This  solution  should  be  added  to  the  media  just 
before  use  in  the  proportion  of  about  ^  cc.  to  5  cc.  of  medium. 

If  litmus  be  added  to  the  milk  before  sterilization,  it  is  apt  to  be 
browned  or  decolorized,  so  that  it  is  better  to  sterilize  the  two  sepa- 
rately and  pour  them  together  subsequently.  It  is  said  that  lac- 
moid  is  never  thus  changed,  and  many  workers  prefer  it  to  litmus  on 
that  account. 

Petruschky's  Whey. — In  order  to  differentiate  between  acid  and 
alkali  producers  among  the  bacteria,  Petruschky  has  recommended  a 
neutral  whey  colored  with  litmus.  It  is  made  as  follows: 

To  a  liter  of  fresh  skimmed  milk  i  liter  of  water  is  added.  The 
mixture  is  violently  shaken.  About  10  cc.  are  taken  out  as  a  sample 
to  determine  how  much  hydrochloric  acid  must  be  added  to  produce 
coagulation  of  the  milk,  and,  having  determined  the  least  quantity 
required  for  the  whole  bulk,  it  is  added.  After  coagulation  the  whey 
is  filtered  off,  exactly  neutralized,  and  boiled.  After  boiling  it  is 
found  clouded  and  acid  in  reaction.  It  is  therefore  filtered  again, 
and  again  neutralized.  Litmus  is  finally  added  to  the  neutral  liquid, 
so  that  it  has  a  violet  color,  changed  to  blue  or  red  by  alkalies  or  acids. 

Peptone  Solution,  or  Dunham's  solution,  is  a  perfectly  clear, 
colorless  solution,  made  as  follows: 

Sodium  chlorid o  •  5 

Witte's  dried  peptone i  .o 

Water 100 .  o 

Boil  until  the  ingredients  dissolve;  filter,  fill  into  tubes  and  sterilize. 

*  "Elements  of  Water  Bacteriology,"  John  Wiley  &  Sons,  New  York,  1904,  p. 
126. 


2OO  Cultivation  of  Micro-organisms 

It  was  for  a  long  time  used  for  the  detection  of  indol.  Garini* 
found  that  many  of  the  peptones  upon  the  market  were  impure,  and 
on  this  account  failed  to  show  the  inck>l  reaction  in  cultures  of  bac- 
teria known  to  produce  it.  He  recommends  testing  the  peptone  to 
be  employed  by  the  use  of  the  biuret  reaction.  The  reagent  em- 
ployed is  Fehling's  copper  solution,  with  which  pure  peptone  strikes 
a  violet  color  not  destroyed  upon  boiling,  while  impure  peptone  gives 
a  red  or  reddish-yellow  precipitate.  Both  the  peptone  and  copper 
solutions  should  be  in  a  dilute  form  to  make  successful  tests. 

The  addition  of  4  cc.  of  the  following  solution — • 

Rosolic  acid 0.5 

Eighty  per  cent,  alcohol 100 .  o 

makes  the  peptone  solution  a  reagent  for  the  detection  of  acids  and 
alkalies.  The  solution  is  of  a  pale  rose  color.  If  the  organisms  cul- 
tivated produce  acids,  the  color  fades;  if  alkalies,  it  intensifies.  As 
the  color  of  rosolic  acid  is  destroyed  by  glucose,  it  cannot  be  used  in 
culture-media  containing  it. 

Theobald  Smithf  has  called  attention  to  the  fact  that  many  bac- 
teria fail  to  grow  in  Dunham's  solution,  and  recommends  that,  for 
the  detection  of  indol,  bouillon  free  of  dextrose  be  used  instead.  All 
bacteria  grow  well  in  it,  and  the  indol  reaction  is  pronounced  in  six- 
teen-hour-old  cultures.  His  method  of  preparation  is  as  follows: 
Beef -infusion,  prepared  either  by  extracting  in  the  cold  or  at  6o°C., 
is  inoculated  in  the  evening  with  a  rich  fluid  culture  of  some  acid- 
producing  bacterium  (Bacillus  coli)  and  placed  in  the  thermostat. 
Early  next  morning  the  infusion,  covered  with  a  thin  layer  of  froth, 
is  boiled,  filtered,  peptone  and  salt  added,  and  the  neutralization  and 
sterilization  carried  on  as  usual. 

This  method  is  subject  to  error,  caused  by  the  presence  in  the  me- 
dium of  indol  produced  by  the  colon  bacillus.  This  can  be  demon- 
strated if  the  tests  for  indol  be  sensitive.  SelterJ  finds  that  the 
method  of  Smith  gives  inferior  results  to  a  simple  culture-medium  con- 
sisting of  water,  90  parts;  Witte's  peptone,  10  parts;  sodium  phos- 
phate, 0.5  part,  and  magnesium  sulphate,  o.i  part. 

Other  culture-media  employed  for  special  purposes  will  be  men- 
tioned as  occasion  arises. 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  xm,  p.  790. 

f  "Journal  of  Exp.  Medicine,"  Sept..  5,  1897,  vi,  p.  546. 

|  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Orig.  LI,  p.  465. 


CHAPTER  VIII 
CULTURES,  AND  THEIR  STUDY 

THE  purposes  for  which  culture-media  are  prepared  are  numerous. 
Through  their  aid  it  is  possible  to  isolate  the  micro-organisms,  to  keep 
them  in  healthy  growth  for  considerable  lengths  of  time,  during 
which  their  biologic  peculiarities  can  be  observed  and  their  metabolic 
products  collected,  and  to  introduce  them  free  from  contamination 
into  the  bodies  of  experiment  animals. 

The  isolation  of  bacteria  was  next  to  impossible  until  the  fluid 
media  of  the  early  observers  were  replaced  by  the  solid  culture-media 
introduced  by  Koch,  and  exceedingly  difficult  until  he  devised  the 
well-known  " plate  cultures." 

A  growth  of  artificially  planted  micro-organisms  is  called  a  culture. 
If  such  a  growth  contains  but  one  kind  of  organism,  it  is  known  as  a 
pure  culture. 

It  has  at  present  become  the  custom  to  use  the  term  "culture" 
rather  loosely,  so  that  it  does  not  always  signify  an  artificially 
planted  growth  of  micro-organisms,  but  may  signify  a  growth  taking 
place  under  natural  conditions;  thus,  the  typhoid  bacillus  is  said  to 
occur  in  "pure  culture"  in  the  spleens  of  patients  dead  of  typhoid 
fever,  because  no  other  bacteria  are  associated  with  it;  and  some- 
times, when  the  tubercle  bacilli  are  very  numerous  and  unmixed 
with  other  bacteria,  in  the  expectorated  fragments  of  cheesy 
matter  from  tuberculosis  pulmonalis,  they  are  said  to  occur  in 
"pure  culture." 

The  culture  manipulations  are  performed  either  with  a  sterilized 
platinum  wire  or  with  a  capillary  pipet  of  glass. 

The  platinum  wire  is  so  limber  that  it  is  scarcely  to  be  recom- 
mended, and  a  wire  composed  of  platinum  and  iridium,  which  is 
elastic  in  quality,  is  to  be  preferred.  The  wires  are  about  5  cm.  in 
length,  of  various  thicknesses  according  to  the  use  for  which  they  are 
employed,  and  are  usually  fused  into  a  thin  glass  rod  about  17  cm.  in 
length.  The  wires  may  be  straight  or  provided  with  a  small  loop 
at  the  end  so  as  to  conveniently  take  up  small  drops  of  fluid.  Heavy 
wires  used  for  securing  diseased  tissue  from  animals  may  be  flattened 
at  the  ends  by  hammering,  and  may  thus  be  fashioned  into  miniature 
knives,  scrapers,  harpoons,  etc.,  as  desired. 

Ravenel  has  invented  a  convenient  form  for  carrying  in  the  pocket. 
It  consists  of  the  platinum  wire 'fastened  in  a  heavier  aluminium  wire 
which  in  turn  fits  into  a  piece  of  glass  tubing.  When  carried  in  the 


2O2  Cultures,  and  their  Study  , 

pocket,  the  position  of  the  platinum  wire  is  reversed  in  the  glass  tub- 
ing and  protected  by  it. 

Immediately  before  and  immediately  after  use,  the  platinum  wire  is 
to  be  sterilized  by  heating  to  incandescence  in  a  flame,  in  order  that  it 
convey  nothing  undesirable  into  the  culture,  and  in  order  that  it 
scatter  no  micro-organisms  about  the  laboratory. 


Fig.  47. — Platinum  needles  for  transferring  bacteria;  made  from  No.  27  platinum 
wire  inserted  in  glass  rods. 

Capillary  glass  tubes  are  employed  by  the  French  for  many  of  the 
manipulations.  They  are  made  of  M-  or  %-inch  glass  tubing  cut 
into  25  cm.  lengths,  heated  at  the  center,  and  drawn  out  to  capillary 
ends  about  5  cm.  long.  They  are  sealed  at  one  end  and  plugged  with 
cotton  at  the  other,  and  a  number  of  them,  prepared  at  the  same  time, 
sterilized.  They  can  be  used  for  all  the  purposes  for  which  the 


Fig.  48. — Ravenel's  platinum  wires  for  bacteriologic  use. 

platinum  wire  is  employed,  and  in  addition  can  be  used  as  con- 
tainers for  small  quantities  of  fluids  sealed  in  them.  When  about  to 
use  such  a  tube,  its  sealed  capillary  end  should  be  broken  off  with 
forceps,  and  the  tube  sterilized  by  flaming. 

Technic  of  Culture  Manipulation. — Containers  of  stored  culture- 
media  should  be  kept  in  an  upright  position,  that  the  cotton  stoppers 


Fig.  49. — Capillary  glass  tubes,  a,  Pipette  for  ordinary  manipulations;  b, 
constricted  pipette  in  which  small  quantities  of  cultures,  etc.,  can  be  sealed  by 
fusing  the  glass;  c  bulbous  pipette  in  which  larger  quantities  of  fluids  may  be 
sealed. 

are  not  moistened  or  soiled.  If  moistened  with  the  culture-media, 
molds  whose  spores  fall  upon  the  surface  of  the  stoppers  may  grad- 
ually work  their  mycelial  threads  between  the  fibers  until  they  ap- 
pear upon  their  inner  surface  and  drop  newly  formed  spores  into  the 
contained  media. 

In  handling  tubes  care  must  be  taken  to  stand  them  up  in  turn- 


,       Technic  of  Culture  Manipulation  203 

biers,  racks,  or  other  contrivances,  and  not  lay  them  upon  the  table 
so  that  the  contents  touch  the  stoppers. 

When  the  cotton  plugs  are  removed  in  order  that  the  contents  of 
the  tubes  or  flasks  maybe  inoculated  or  otherwise  manipulated,  the 
removal  and  replacement  should  be  done  as  quickly  as  convenient, 
and  the  mouth  of  the  tube  should  be  flamed  before  removal.  The 
plugs  should  be  held  between  the  fingers,  by  that  part  which  projects 
above  the  glass,  not  laid  upon  the  table,  from  which  dust,  and  in- 
cidentally bacteria,  may  be  taken  up  and  subsequently  dropped  into 
the  medium;  nor  must  they  be  touched  with  the  fingers  at  that  part 
which  enters  the  neck  of  the  container  lest  they  take  up  micro- 
organisms from  the  skin.  The  stoppers  thus  require  careful  con- 
sideration lest  they  become  the  source  of  future  contamination. 

So  soon  as  the  cotton  stopper  is 
removed,  the  medium  is  left  with- 
out protection  from  whatever 
micro-organisms  happen  to  be  in 
the  air,  so  that  it  should  be  re- 
placed as  soon  as  possible,  and 
every  manipulation  requiring  its 
removal  performed  expeditiously. 
During  the  time  the  stopper  is 
withdrawn  it  is  wise  to  hold  the 
tubes  or  other  containers  in  an 
oblique  or  horizontal  position 
that  will  aid  in  excluding  the 
micro-organisms  of  the  air.  Some 
bacteriologists  make  inoculations  pig  so._Method  of  hoidin^ubes 
with  the  tubes  reversed  in  all  during  inoculation, 

cases   in   which   solid  media   are 

employed,  but  it  is  not  necessary.  If  the  tubes  are  held  obliquely, 
the  danger  of  contamination  is  reduced  to  a  minimum.  It  is  well 
to  adopt  some  method  of  handling  the  tubes  that  has  given  satisfac- 
tion to  others  and  is  found  convenient  to  one's  self  and  habitually 
practise  it  until  it  becomes  second  nature  and  can  be  done  without 
thought. 

The  usual  method  of  making  a  transplantation  of  bacteria  from 
culture- tube  to  culture- tube,  is,  in  detail,  as  follows: 

In  order  that  any  bacteria  loosely  scattered  over  the  surface  of  the 
cotton  stopper,  and  upon  the  glass  near  the  mouth  of  the  tube,  may 
be  destroyed  and  prevented  from  entering  the  medium  as  the  stopper 
is  withdrawn,  both  the  tube  containing  the  culture  and  the  fresh 
tube  to  which  it  is  to  be  transferred  should  be  held  for  a  moment  in  a 
flame  and  rolled  from  side  to  side  so  that  all  parts  are  flamed.  The 
cotton  ignites  and  blazes  actively,  but  the  flame  can  be  extinguished 
by  forcibly  blowing  upon  it  and  any  smoldering  remains  extinguished 
by  pinching  with  the  fingers.  The  tubes  are  now  placed  side  by  side 


2O4  Cultures,  and  their  Study 

between  the  thumb  and  upward-directed  palm  of  the  left  hand,  the 
stoppers  toward  the  operator.  The  position  of  the  tubes  should  be 
such  as  to  permit  one  to  see  the  contained  media  without  the  fingers 
being  in  the  way.  The  stopper  of  the  tube  toward  the  left  is  re- 
moved by  a  gentle  twist  and  placed  between  the  index  and  middle 
fingers  of  the  left  hand;  the  stopper  of  the  next  tube  similarly  re- 
moved and  placed  between  the  middle  and  ring  fingers  of  the  same 
hand.  If  three  or  four  tubes  are  to  be  held,  the  third  stopper 
can  be  placed  between  the  ring  and  little  fingers  of  the  left  hand 
and  the  fourth  retained  in  the  right  hand.  The  part  of  each  stopper 
that  enters  the  tube  must  not  be  touched. 

The  necessary  manipulation  is  usually  made  with  the  platinum 
wire,  which  is  sterilized  by  heating  to  incandescence  before  using. 
The  wire  must  not  be  used  while  hot,  but  cools  in  a  moment  or  two. 
The  culture  is  touched,  the  wire  entering  and  exiting  without  touch- 
ing the  tube,  and  the  bacteria  adhering  to  the  wire  are  applied  to  the 
medium  in  the  other  tube,  the  same  care  being  exerted  not  to  have 
the  platinum  wire  touch  the  glass.  After  the  transfer  is  made,  the 
wire  is  made  incandescent  in  the  flame  before  being  returned  to  the 
table  or  stand  made  to  hold  it,  and  the  stoppers  returned  one  after 
the  other,  each  to  its  own  tube,  that  part  entering  the  tube  not  being 
touched.  Each  stopper  is  given  a  twist  as  it  enters  the  mouth  of  the 
tube. 

Modifications  of  these  directions  can  be  made  to  suit  the  differ- 
ent forms  of  containers  used,  but  the  essential  features  must  be 
maintained. 

When  any  manipulation  requires  that  a  tube  or  flask  be  permitted 
to  remain  open  an  unusual  length  of  time,  its  contamination  from  the 
air  can  be  prevented  for  some  minutes  by  heating  its  neck  quite 
hot.  The  air  about  it,  being  heated  by  the  hot  glass,  ascends,  form- 
ing a  current  that  carries  the  bacteria  away  from,  rather  than  into, 
the  receptacle. 

Isolation  of  Bacteria. — Three  principal  methods  are,  at  present, 
employed  for  securing  pure  cultures  of  bacteria.  Before  beginning  a 
description  of  them  it  is  well  to  observe  that  the  peculiarities  of 
certain  pathogenic  micro-organisms  enable  us  to  use  special  means  for 
their  isolation,  and  that  these  general  methods  are  chiefly  useful  for 
the  isolation  of  non-pathogenic  organisms. 

Plate  Cultures. — All  the  methods  depend  upon  the  observation  of 
Koch,  that  when  bacteria  are  equally  distributed  throughout  some 
liquefied  nutrient  medium  that  is  subsequently  solidified  in  a  thin 
layer,  they  grow  in  scattered  groups  or  families,  called  colonies,  dis- 
tinctly isolated  from  one  another  and  susceptible  of  transplantation. 

The  plate  cultures,  as  originally  made  by  Koch,  require  con- 
siderable apparatus,  and  of  late  years  have  given  place  to  the  more 
simple  and  ready  methods.  So  great  is  their  historic  interest,  how- 


Plate  Cultures 


205 


ever,  that  it  would  be  a  great  omission  not  to  describe  the  original 
method  in  detail. 

Apparatus. — Half  a  dozen  glass  plates,  measuring  about  6  by  4  inches,  free 
from  bubbles  and  scratches  and  ground  at  the  edges,  are  carefully  cleaned,  placed 
in  a  sheet-iron  box  made  to  receive  them,  and  sterilized  in  the  hot-air  closet. 
The  box  is  kept  tightly  closed,  and  in  it  the  sterilized  plates  can  be  kept 
indefinitely  before  use. 

A  moist  chamber,  or  double  dish,  about  10  inches  in  diameter  and  3  inches 
deep,  the  upper  half  being  just  enough  larger  than  the  lower  to  allow  it  to  close 
over  it,  is  carefully  washed.  A  sheet  of  bibulous  paper  is  placed  in  the  bottom, 
so  that  some  moisture  can  be  retained,  and  a  i  :  1000  bichlorid  of  mercury  solu- 
tion poured  in  and  brought  in  contact  with  the  sides,  top,  and  bottom  by  turning 
the  dish  in  all  directions.  The  solution  is  emptied  out,  and  the  dish,  which  is 
kept  closed,  is  ready  for  use. 

A  leveling  apparatus  is  required.  It  consists  of  a  wooden  tripod  with  ad- 
justable screws,  and  a  glass  dish  covered  by  a  flat  plate  of  glass  upon  which  a  low 
bell-jar  stands.  The  glass  dish  is  filled  with  broken  ice  and  water,  covered 
with  the  glass  plate,  and  then  exactly 
leveled  by  adjusting  the  screws  under  the 
legs  of  the  tripod.  When  level,  the  cover 
is  placed  upon  it,  and  it  is  ready  for  use. 
Method. — A  sterile  platinum  loop  is 
dipped  into  the  material  to  be  examined, 
a  small  quantity  secured,  and  stirred  about 
so  as  to  distribute  it  evenly  throughout 
the  contents  of  a  tube  of  melted  gelatin. 
If  the  material  under  examination  be  very 
rich  in  bacteria,  one  loopful  may  contain  a 
million  individuals,  which,  if  spread  out 
in  a  thin  layer,  would  develop  so  many 
colonies  that  it  would  be  impossible  to  sec 
any  one  clearly;  hence  further  dilation  be- 
comes necessary.  From  the  first  tube, 
therefore,  a  loopful  of  gelatin  is  carried  to 
a  second  and  stirred  well,  so  as  to  distribute 
the  organisms  evenly  throughout  its  con- 
tents. In  this  tube  we  may  have  no  more  than  ten  thousand  organisms,  and  if 
the  same  method  of  dilution  be  used  again,  the  third  tube  may  have  only  a  few 
hundred,  and  a  fourth  only  a  few  dozen  colonies. 

After  the  tubes  are  tnus  inoculated,  one  of  the  sterile  glass  plates  is  caught  by 
its  edges,  removed  from  the  iron  box,  and  placed  beneath  the  bell-glass  upon  the 
cold  plate  covering  the  ice- water  of  the  leveling  apparatus.  The  plug  of  cotton 
closing  the  mouth  of  tube  No.  i  is  removed,  and  to  prevent  contamination  during 
the  outflow  of  the  gelatin  the  mouth  of  the  tube  is  held  in  the  flame  of  a  Bunsen 
burner  for  a  moment  or  two.  The  gelatin  is  then  cautiously  poured  out  upon  the 
plate,  the  mouth  of  the  tube,  as  well  as  the  plate,  being  covered  by  the  bell-glass 
to  prevent  contamination  by  germs  in  the  air.  The  apparatus  being  level,  the 
gelatin  spreads  out  in  an  even,  thin  layer,  and,  the  plate  being  cooled  by  the  ice 

beneath,  it  immediately  solidifies,  and  in  a  few 
moments  can  be  removed  to  the  moist  cham- 
ber prepared  to  receive  it.  As  soon  as  plate 
No.  i  is  prepared,  the  contents  of  tube  No.  2 
are  poured  upon  plate  No.  2,  allowed  to  spread 
out  and  solidify,  and  then  superimposed  on 
plate  No.  i  in  the  moist  chamber,  being  sepa- 
rated from  the  plate  already  in  the  chamber 
by  small  glass  benches  made  for  the  purpose  and  previously  sterilized.  After 
the  contents  of  all  the  tubes  are  thus  distributed,  the  moist  chamber  and 
its  contents  are  stood  away  to  permit  the  bacteria  to  grow.  Where  each 
organism  falls  a  colony  develops,  and  the  success  of  the  whole  method  depends 
upon  the  isolation  of  a  colony  and  its  transfer  to  a  tube  of  new  sterile  culture- 
media,  where  it  can  grow  unmixed  and  undisturbed. 

From  the  description  it  must  be  evident  that  only  those  culture-media  that 


Fig.  51. — Complete  leveling  ap- 
paratus for  pouring  plate  cultures, 
as  taught  by  Koch. 


Fig.  52. — Glass  bench. 


2O6 


Cultures,  and  their  Study 


can  be  melted  and  solidified  at  will  can  be  used  for  plate  cultures — viz.,  gelatin, 
agar-agar,  and  glycerin  agar-agar.  Blood-serum  and  Loflfler's  mixture  are  en- 
tirely inappropriate. 

The  chief  drawbacks  to  this  excellent  method  are  the  cumbersome 
apparatus  required  and  the  comparative  impossibility  of  making 
plate  cultures,  as  is  often  desirable,  in  the  clinic,  at  the  bedside,  or 
elsewhere  than  in  the  laboratory.  The  method  therefore  soon  under- 
went modifications,  the  most  important  being  that  of  Petri,  who  in- 
vented special  dishes  to  be  used  instead  of  plates. 

Petri's  Dishes. — These  are  glass  dishes,  about  4  inches  in  diameter 
and  %  inch  deep,  with  accurately  fitting  lids.  They  were  first 


Fig-  53- — Petri  dish  for  making  plate  cultures. 

recommended  by  Petri*  and  greatly  simplify  bacteriologic  technic 
by  dispensing  with  the  plates  and  plate-boxes,  the  moist  chambers 
and  benches,  and  usually  with  the  levelling  apparatus  of  Koch, 
though  this  is  still  employed  in  some  laboratories,  and  must  always 
be  employed  when  an  even  distribution  of  the  colonies  is  necessary 
in  order  that  they  can  be  accurately  counted. 

The  method  of  using  the  Petri  dishes  is  very  simple.     They  are 
carefully  cleaned,  polished,  closed  and  sterilized  by  hot   air,  care 


Fig.  54. — Petri  dish  forceps. 

being  taken  that  they  are  placed  in  the  hot-air  closet  right  side  up, 
and  after  sterilization  are  kept  covered  and  in  that  position.  They 
should  be  sterilized  immediately  before  using,  or  if  they  must  be 
kept  for  a  time  should  be  wrapped  in  tissue  paper  and  then  sterilized. 
The  tissue  paper  protects  the  accidental  entrance  of  dust  between 
dish  and  lid,  keeps  the  dish  closed,  and  need  not  be  removed 
until  the  last  moment  before  using. 

Time  can  be.  saved  by  sterilizing  the  dish  and  cover  in  the  direct 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1887,  i,  No.  i,  p.  279. 


Esmarch's  Tubes  207 

flame,  instead  of  in  the  hot-air  closet,  special  forceps  adapted  to 
holding  them  having  been  devised  by  Rosenberger.  * 

The  dilution  of  the  material  under  examination  is  made  with  gela- 
tin or  agar-agar  tubes  in  the  manner  above  described,  the  plug  is 
removed,  the  mouth  of  the  tube  cautiously  held  for  a  moment  in  the 
flame,  and  the  contents  poured  into  one  of  the  sterile  dishes,  whose 
lid  is  just  sufficiently  elevated  to  permit  the  mouth  of  the  tube  to 
enter.  The  gelatin  is  spread  over  the  bottom  of  the  dish  in  an  even 
layer,  allowed  to  solidify,  labeled,  inverted,  so  that  the  water  of  con- 
densation may  not  drop  from  the  lid  upon  the  culture  film  and  spoil 
the  cultures,  and  stood  away  for  the  colonies  to  develop. 


Fig.  55- — Esmarch  tube  on  block  of  ice  (redrawn  after  Abbott). 

To  overcome  the  difficulty  of  excessive  water  of  condensation  Hill 
has  introduced  lids  made  of  porous  clay,  by  which  the  moisture  is 
absorbed.  These  can  be  obtained  from  most  laboratory  purveyors. 

Among  the  other  advantages  of  the  Petri  dish  is  the  convenience 
with  which  colonies  can  be  studied  with  a  low-power  lens.  To  do 
this  with  the  Koch  plates  meant  to  remove  them  from  the  sterile 
chamber  to  the  stage  of  a  microscope  and  so  expose  them  to  the  air, 
and  to  contamination,  but  to  examine  colonies  in  the  Petri  dish,  one 
simply  examines  through  the  thin  glass  of  the  bottom  dish  without 
any  exposure  to  contaminating  organisms. 

Esmarch's  Tubes. — This  method,  devised  by  Esmarch,  converts  the  wall  of 
the  test-tube  into  the  plate  and  dispenses  with  all  other  apparatus.  The  tubes, 
which  are  inoculated  and  in  which  the  dilutions  are  made,  should  contain  less 
than  half  the  usual  amount  of  gelatin  or  agar-agar.  After  inoculation  the  cotton 
plugs  are  pushed  into  the  tubes  until  even  with  their  mouths,  and  then  covered 
with  a  rubber  cap,  which  protects  them  from  wetting.  A  groove  is  next  cut  in  a 
block  of  ice,  and  the  tube,  held  almost  horizontally,  is  rolled  in  this  until  the  entire 
surface  of  the  glass  is  covered  with  a  thin  layer  of  the  soldified  medium.  Thus 
the  wall  of  the  tube  becomes  the  plate  upon  which  the  colonies  develop. 

In  carrying  out  Esmarch's  method,  the  tube  must  not  contain  too  much  of 
the  culture  medium,  or  it  cannot  be  rolled  into  an  even  layer;  the  contents  should 
not  touch  the  cotton  plug,  lest  it  be  glued  to  the  glass  and  its  subsequent  useful- 
ness injured,  and  no  water  must  be  admitted  from  the  melted  ice. 

*  "Phila.  Med.  Jour.,"  Oct.  20,  1900,  vol.  vi,  No.  16,  p.  760. 


208 


Cultures,  and  their  Study 


Colonies. — The  progeny  of  each  bacterium  form  a  mass  which  is 
known  as  a  colony.  When  these  are  separated  from  one  another, 
each  is  spoken  of  as  a  single  colony,  and  different  characteristics 
belonging  to  different  micro-organisms  enable  us  at  times  to  recognize 
by  macroscopic  and  microscopic  study  of  the  colony  the  particular 
kind  of  micro-organism  from  which  it  has  grown.  The  illustrations 
show  the  various  types  of  colonies  and  the  legends  the  terms  used 
in  describing  them. 

Growing  colonies  should  be  observed  from  day  to  day,  as  it  not 
infrequently  happens  that  unexpected  changes,  such  as  pigmenta- 


Fig.  56. — Types  of  colonies:  a,  Cochleate  (B.  coli,  abnormal  form) ;  b,  conglom- 
erate (B.  zopfii);  c,  ameboid  (B.  vulgatus) ;  d,  filamentous  (Frost). 

tion  and  liquefaction,  develop  after  the  colony  is  several  days  old 
and  indeed  sometimes  not  until  much  later.  Again,  many  colonies 
make  their  first  appearance  as  minute,  sharply  circumscribed  points, 
and  later  spread  upon  the  surface  of  the  culture-medium,  either  in 
the  form  of  a  thin,  homogeneous  layer  or  a  filamentous  cluster.  It 
is  particularly  important  that  in  describing  new  species  of  bac- 
teria an  account  of  the  appearance  of  the  colonies  from  day  to  day, 
comparing  all  of  their  variations  for  at  least  two  weeks,  should 
be  included. 


Tig.  57- — Surface  elevations  of  growths:  a,  Flat;  b,  raised;  c,  convex;  d,  pulvi- 
nate;  e,  capitate;/,  umbilicate;  g,  umbonate  (Frost). 

Pure  Cultures. — Single  colonies  also  subserve  a  second  very  im- 
portant purpose,  that  of  enabling  us  to  secure  pure  cultures  of  bacteria 
from  a  mixture.  For  this  purpose  an  isolated  colony  is  selected  and 
carefully  examined  to  see  that  it  is  single  and  not  a  mixture  of  two 
closely  approximated  colonies  of  different  kinds,  and  then  trans- 
planted to  a  tube  of  an  appropriate  culture-medium.  If  the  colonies 
are  few  and  of  good  size,  each  is  picked  up  with  a  sterile  platinum 
wire  and  transplanted  to  a  tube  of  appropriate  culture-medium. 
If,  however,  the  colonies  are  numerous,  of  small  size,  and  close  to- 


The  Gelatin  Puncture  or  "Stab"  Culture 


209 


gether,  it  may  be  necessary  to  do  it  under  a  dissecting  microscope 
or  even  a  low  power  of  the  ordinary  bacteriologic  microscope. 
This  operation  of  transplantation  is  familiarly  known  as  fishing. 

Fishing. — It  requires  considerable  practice  and  skill  to  fish  suc- 
cessfully, and  the  student  should  early  begin  to  practise  it.  The 
colony  to  be  transplanted,  selected  because  of  its  isolation,  its  typical 
appearance,  and  convenient  position  on  the  plate,  is  brought  to  the 
center  of  the  field  and  the  plate  firmly  held  in  position  with  the  left 
hand.  A  sterile  platinum  wire  is  held  in  the  right  hand,  the  little 
finger,  comfortably  fixed  upon  the  stage  of  the  microscope,  being  used 
to  support  the  hand.  As  the  operator  looks  into  the  microscope  the 
point  of  the  platinum  wire  is  carefully  brought  into  the  field  of  vision 
without  touching  either  the  lens  of  the  microscope  or  any  part  of  the 
plate  beneath.  Of  course,  the  wire  and  the  colony  cannot  be  simul- 


Fig.  58. — Microscopic  structure  of  colonies:  i,  Areolate;  2,  grumose;  3, 
moruloid;  4,  clouded;  5,  gyrose;  6,  marmorated;  7,  reticulate,  8,  repand;  9,  lobate; 
10,  erose;  n,  auriculate;  12,  lacerate;  13,  fimbricate;  14,  ciliate  (Frost). 

taneously  focussed  upon.  When  the  colony  is  distinctly  seen  the 
platinum  wire  appears  as  a  shadow,  but  the  endeavor  should  be  to 
make  the  end  of  the  shadow  which  corresponds  to  the  point  of  the 
wire  appear  exactly  over  the  colony.  It  is  then  gradually  depressed 
until  it  touches  the  colony  and  can  be  seen  to  break  up  and  remove 
some  of  its  substance;  or  should  the  colony  be  tough  and  coherent,  to 
tear  it  away  from  the  culture-medium.  It  requires  almost  as  much 
skill  to  withdraw  the  wire  from  the  colony  without  touching  anything 
as  to  successfully  approach  the  colony  in  the  first  place.  The 
bacterial  mass  adhering  to  the  wire  is  now  spread  upon  the  surface 
of  agar-agar  or  stabbed  in  gelatin  or  stirred  in  fluid  medium,  as  the 
case  may  be.  The  higher  the  magnification  under  which  this  opera- 
tion is  done,  the  more  difficult  it  is.  Therefore  only  low-power 
lenses  should  be  employed. 

The  Gelatin  Puncture  or  "Stab"  Culture.— To  make  satisfactory 
puncture  cultures,  the  gelatin  must  be  firm  but  not  old  or  dry. 
Should  the  gelatin  be  soft  and  semi-fluid  at  the  time  the  puncture  is 
made,  the  bacteria  diffuse  themselves  and  the  typical  appearance 
of  the  growth  may  be  masked.  On  the  other  hand,  if  the  gelatin  be 
old,  dry,  or  retracted,  it  is  very  apt  to  crack  after  the  culture  has  been 
14 


210 


Cultures,  and  their  Study 


made  and  thus  entirely  destroy  the  characteristics  of  the  growth. 
The  wire  used  in  the  operation  should  be  perfectly  straight,  and  the 
puncture  should  be  made  from  the  center  of  the  surface  directly 
down  to  the  bottom  of  the  tube  and  then  withdrawn,  so  that  a 
simple  puncture  is  made.  The  appearances  presented  as  the  growth 
progresses  are  subject  to  striking  variations  according  to  the  lique- 
fying or  non-liquefying  tendency  of  the  micro-organisms.  Various 
types  of  gelatin  cultures  are  shown  in  the  accompanying  diagrams, 


Fig.  59. — Types  of  growth  in  stab  cultures.  A,  Non-liquefying:  i,  Filiform 
'(B.  coli);  2,  beaded  (Str.  pyogenes);  3,  echinate  (Bact.  acidi-lactici) ;  4,  villous 
(Bact.  murisepticum);  5,  arborescent  (B.  mycoides).  B,  Liquefying:  6,  Crateri- 
form  (B.  vulgare,  24  hours);  7,  napiform  (B.  subtilis,  48  hours);  8,  infundibuli- 
form  (B.  prodigiosus) ;  9,  saccate  (Msp.  finkleri);  10,  stratiform  (Ps.  fluorescens) 
(Frost). 

and  it  is  rather  important  that  the  student  should  familiarize  himself 
with  the  terms  by  which  these  different  growths  are  described,  in 
order  that  uniformity  of  description  may  be  maintained.  Gelatin 
cultures  may  not  be  kept  in  the  incubating  oven,  as  the  medium 
liquefies  at  such  temperatures.  On  the  other  hand,  they  must  not  be 
kept  where  the  temperature  is  too  low,  else  the  bacterial  growth 
may  be  retarded.  The  temperature  of  a  comfortably  heated  room, 
not  subject  to  excessive  variations,  such  as  are  caused  by  steam 
heat  and  the  burning  of  gas,  etc.,  is  about  the  most  appropriate. 
Like  the"  colonies,  the  cultures  must  be  carefully  examined  from  day 
to  day,  as  it  not  infrequently  happens  that  a  growth  which  shows  no 


Cultures  upon  Potato 


211 


signs  of  liquefaction  to-day  may  begin  to  liquefy  to-morrow  or  a  week 
hence,  or  even  as  late  as  two  weeks  hence. 

The  Agar-agar  Culture. — In  most  cases,  the  culture  is  planted  by 
a  simple  stroke  made  from  the  bottom  of  the  tube  in  which  the  agar- 
agar  has  been  obliquely  solidified,  and  where  it  is  fresh  and  moist, 
to  the  upper  part,  where  it  is  thin  and  dry.  In  addition  to  this,  it  is 
advisable  to  make  a  puncture  from  the  center  of  the  oblique  surface 
to  the  bottom  of  the  tube.  This  enables  us  to  tell  whether  the  bacte- 
ria can  grow  as  readily  below  the  surface  as  above.  Some  workers 
always  make  a  zigzag  stroke  upon  the  surface  of  the  agar-agar.  This 
does  not  seem  to  have  any  particular  advantage  except  in  cases  where 
it  is  desired  to  scatter  the  transplanted  organisms  as  much  as  possible, 
in  order  that  a  large  bacterial  mass  may  be  secured. 

The  growth  upon  agar-agar  is  in  many  ways  less  characteristic 
than  in  gelatin,  but  as  the  medium  does  not  liquefy  except  at  a  high 
temperature  (ioo°C.),  it  has  the  advantage  that  cultures  may  be 


Fig.  60.— Types  of  streak  cultures:  i,  Filiform  (B.  coli);  2,  echinulate  (Bact. 
acidi-lactici) ;  3,  beaded  (Str.  pyogenes);  4,  effuse  (B.  vulgaris);  5,  arborescent 
(B.  mycoides)  (Frost). 

kept  in  the  incubating  oven.  The  colorless  or  almost  colorless  con- 
dition of  the  preparation  also  aids  in  the  detection  of  chromogenesis. 

The  growth  may  be  filamentous,  or  simply  a  smooth,  shining  band. 
Occasionally  the  bacterium  does  not  grow  upon  agar-agar  unless 
glycerin  be  added  (tubercle  bacillus);  sometimes  it  will  not  grow 
even  then  (gonococcus). 

Cultures  upon  Blood-serum. — Bacteria  are  planted  upon  coagu- 
lated blood  serum  and  blood-serum  preparations  as  upon  agar-agar. 

Blood-serum  is  liquefied  by  some  bacteria,  but  the  majority  of 
organisms  have  no  characteristic  reaction  upon  it.  A  few,  as  the 
bacillus  of  diphtheria,  are,  however,  characterized  by  rapid  develop- 
ment at  given  temperatures. 

Cultures  upon  Potato. — These  are  made  by  simply  stroking  the 
surface  of  the  culture-medium,  the  density  and  opacity  of  the 
potato  making  it  impracticable  to  puncture  it. 

Most  bacteria  produce  smooth,  shining,  irregularly  extending 
growths  upon  potato,  that  may  show  characteristic  colors. 


212  Cultures,  and  their  Study 

Cultures  in  Fluid  Media. — Here,  as  has  already  been  stated, 
transplantation  consists  in  simply  stirring  in  the  bacteria  so  as  to 
distribute  them  fairly  well  throughout  the  medium. 

In  milk  and  litmus  milk  one  should  observe  change  in  color  from 
the  occurrence  of  acid  or  alkali  production,  coagulation,  gelatiniza- 
tion,  and  digestion  of  the  coagulum. 

Adhesion  Preparations. — Sometimes  it  is  desirable  to  preserve 
an  entire  colony  as  a  permanent  microscopic  specimen.  To  do  this 
a  perfectly  clean  cover-glass,  not  too  large  in  size,  is  momentarily 
warmed,  then  carefully  laid  upon  the  surface  of  the  gelatin  or  agar- 
agar  containing  the  colonies.  Sufficient  pressure  is  applied  to  the 
surface  of  the  glass  to  exclude  bubbles,  but  not  to  destroy  the  integ- 
rity of  the  colony.  The  cover  is  gently  raised  by  one  edge,  and  if 
successful  the  whole  colony  or  a  number  of  colonies,  as  the  case  may 
be,  will  be  found  adhering  to  it.  It  is  treated  exactly  as  any  other 
cover-glass  preparation — dried,  fixed,  stained,  mounted,  and  kept  as 
a  permanent  specimen.  It  is  called  an  adhesion  preparation — 
"  Klatschpraparat" 

Special  Methods  of  Securing  Pure  Cultures. — Pure  cultures  from 
single  colonies  may  also  be  secured  by  a  very  simple  manipulation 
suggested  by  BantL*  The  inoculation  is  made  into  the  water  of 
condensation  at  the  bottom  of  an  agar-agar  tube,  without  touching 
the  surface.  The  tube  is  then  inclined  so  that  the  water  flows  over 
the  agar,  after  which  it  is  stood  away  in  the  vertical  position.  Colo- 
nies will  grow  where  bacteria  have  been  floated  upon  the  agar- 
agar,  and  may  be  picked  up  later  in  the  same  manner  as  from  a 
plate. 

When  the  bacterium  to  be  isolated  (gonococcus,  etc.)  will  not  grow 
upon  media  capable  of  alternate  solidification  and  liquefaction, 
the  blood-serum,  potato,  or  other  medium  may  be  repeatedly  stroked 
with  the  platinum  wire  dipped  in  the  material  to  be  investigated. 
Where  the  first  strokes  were  made,  confluent  impure  cultures  occur; 
but  as  the  wire  became  freer  of  organisms  by  repeated  contact  with 
the  medium,  the  colonies  become  scattered  and  can  be  studied  and 
transplanted. 

In  some  cases  pure  cultures  may  be  most  satisfactorily  secured 
by  animal  inoculation.  For  example,  when  the  tubercle  bacillus 
is  to  be  isolated  from  milk  or  urine  which  contains  bacteria  that 
would  outgrow  the  slow-developing  tubercle  bacillus,  it  is  better 
to  inject  the  fluid  into  the  abdominal  cavity  of  a  guinea-pig,  await 
the  development  of  tuberculosis  in  the  animal,  and  then  seek  to 
secure  pure  cultures  of  the  bacillus  from  the  unmixed  infectious 
lesions. 

In  other  cases,  as  when  it  is  desired  to  isolate  Micrococcus  tetrag- 
enus,  the  pneumococcus,  and  other  bacteria  that  pervade  the  blood, 
it  is  easier  to  inoculate  the  animal  most  susceptible  to  the  infection 
*"Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1895,  xvn,  No.  16. 


Microscopic  Study  of  Cultures 


213 


and  recover  it  from  the  blood  or  organs,  than  to  plate  it  out  and 
search  for  the  colony  among  many  others  similar  to  it. 

Microscopic  Study  of  Cultures. — Some  attention  has  been  given 
to  the  preparation  of  microtome  sections  of  gelatin  cultures,  though 


Fig.  61. — Modern  incubating  oven. 

not  much  practical  value  has  come  of  it.  It  can  be  done  by  warming 
the  glass  of  the  tube  sufficiently  to  permit  the  gelatin  containing  the 
growth  to  be  removed  in  a  lump  and  placed  in  Miiller's  fluid  (bichro- 
mate of  potassium  2-2.5,  sulphate  of  sodium  i,  water  100),  where  it 


214  Cultures,  and  their  Study 

is  hardened.  When  quite  firm  it  is  washed  in  water,  passed  through 
alcohols  ascending  in  strength  from  50  to  100  per  cent.,  embedded 
in  celloidin,  cut  wet,  and  stained  like  a  section  of  tissue. 

Winkler*  accomplishes  the  same  end  by  boring  a  hole  in  a  block 
of  paraffin  with  the  smallest  size  cork-borer,  soaks  the  block  in  bi- 
chlorid  solution  for  an  hour,  pours  liquid  gelatin  into  the  cavity, 
allows  it  to  solidify,  inoculates  it  by  the  customary  puncture  of  the 
platinum  wire,  allows  it  to  develop  sufficiently,  and  when  ready 
cuts  the  sections  under  alcohol,  subsequently  staining  them  with 
much  diluted  carbol-fuchsin. 

Museum.  Culture  Preparations. — Neat  museum  specimens  of  plate 
and  puncture  cultures  in  gelatin  can  be  made  by  simultaneously 
killing  the  micro-organisms  and  fixing  the  gelatin  with  formaldehyd, 
which  can  either  be  sprayed  upon  the  gelatin  or  applied  in  dilute 
solution.  As  gelatin  fixed  in  formaldehyd  cannot  subsequently  be 
liquefied,  such  preparations  will  last  a  long  time. 

Standardizing  Freshly  Isolated  Cultures. — This  is  a  matter  of 
some  importance,  as  in  bringing  bacteria  into  the  new  environment 
of  artificial  cultivation  their  biologic  peculiarities  are  temporarily 
altered,  and  it  takes  some  time  for  them  to  recover  themselves. 
While  the  appearances  of  the  freshly  isolated  organism  should  be 
carefully  noted,  too  much  stress  should  not  be  laid  upon  them,  and 
before  beginning  the  systematic  study  of  any  new  organism  it  should 
be  made  to  grow  for  several  successive  generations  upon  two  or 
three  of  the  most  important  culture  media.  Its  saprophytic  exist- 
ence being  thus  established,  the  characteristics  manifested  become 
the  permanent  peculiarities  of  the  species. 

*  " Fortschritte  der  Medicin,"  1893,  Bd.  xi,  No.  22. 


CHAPTER  IX 
THE  CULTIVATION  OF  ANAEROBIC  ORGANISMS 

THE  presence  of  uncombined  oxygen  in  ordinary  cultures  inhibits 
the  development  of  anaerobic  bacteria.  When  such  are  to  be  culti- 
vated, it  therefore  becomes  necessary  to  utilize  special  apparatus 
or  adopt  physical  or  chemic  methods  for  the  exclusion  of  the  air. 
Many  methods  have  been  suggested  for  the  purpose,  an  excellent 
review  of  which  has  recently  been  published  by  Hunziker,*  who 
divides  them  as  follows,  according  to  the  principle  by  which  the 
anaerobiosis  is  brought  about: 

1.  By  the  formation  of  a  vacuum. 

2.  By  the  displacement  of  the  air  by  inert  gases. 

3.  By  the  absorption  of  the  oxygen. 

4.  By  the  reduction  of  the  oxygen. 

5.  By  the  exclusion  of  atmospheric  air  by  means  of  various 

physical  principles  and  mechanical  devices. 

6.  By  the  combined  application  of  any  two  or  more  of  the 
above  principles. 

This  classification  makes  such  an  excellent  foundation  for  the 
description  of  the  methods  that  it  has  been  unhesitatingly  adopted. 

1.  Withdrawal  of  the  Air  and  the  Formation  of  a  Vacuum. — This 
method  was  first  suggested  by  Pasteur  and  was  later  modified  by 
Roux,  Gruber,  Zupinski,  Novy,  and  others.     It  is  now  rarely  em- 
ployed.    The  appropriate  container,  whether  a  tube,  flask,  or  some 
special  device  such  as  the  Novy  jar,  receives  the  culture,  and  then 
has  the  air  removed  by  a  vacuum  pump,  the  tube  either  being  sealed 
in  a  flame  or  closed  by  a  stop-cock. 

2.  Displacement  of  the  Air  by  Inert  Gases. — This  method  is 
decidedly  preferable  to  the  preceding,  as  it  leaves  no  vacuum.     It 
is  easier  to  displace  the  oxygen  than  to  withdraw  it,  and  any  appa- 
ratus permitting  a  combination  of  both  features,  as  that  designed  by 
Ravenel,f  from  which  the  air  can  be  sucked  by  a  pump,  to  be  later 
replaced  by  hydrogen,  can  be  viewed  with  favor. 

The  most  simple  apparatus  of  the  kind  was  suggested  by  Frankel 
who  inoculated  a  culture-tube  of  melted  gelatin  or  agar-agar,  solidi- 
fied it  upon  the  wall  of  the  tube,  as  suggested  by  Esmarch,  sub- 

*  "Journal  of  Applied  Microscopy  and  Laboratory  Methods,"  March,  April  and 
May,  1902;  vol.  v,Nos.  3, 4,  and  5. 

t" Bacteria  of  the  Soil,"  "Memoirs  of  the  National  Academy  of  Sciences," 
First  Memoir,  1896. 

215 


2l6 


The  Cultivation  of  Anaerobic  Organisms 


stituted  for  the  cotton  stopper  a  sterile  rubber  cork  containing  a 
long  entrance  and  short  exit  tube  of  glass,  passed  hydrogen  through 
the  tube  until  the  oxygen  had  been  entirely  removed,  then  sealed  the 
ends  in  a  flame.  In  this  tube  the  growth  of  superficial  and  deep 
colonies  can  be  observed.  Hansen  and  Liborius  constructed  special 


Fig.  62. — Novy's  jars  for  anaerobic  cultures. 


Fig.  63. — Franker  s 
method  of  making 
anaerobic  cultures. 


Fig.  64. — Liborius'  tube 
for  anaerobic  cultures. 


tubes  by  fusing  a  small  glass  tube  into  the  wall  of  a  culture-tube, 
and  narrowing  the  upper  part  of  the  tube  in  a  flame.  After  inocu- 
lation, hydrogen  is  passed  into  the  small  tube  and  permitted  to  es- 
cape through  the  mouth  of  the  large  tube  until  the  air  is  entirely 
replaced",  after  which  both  tubes  are  sealed  in  a  flame. 

Instead  of  having  a  special  apparatus  for  each  culture,  it  is  far 


The  Absorption  of  the  Atmospheric  Oxygen  217 

better  to  adapt  the  principle  to  some  larger  piece  of  apparatus  that 
can  contain  a  number  of  tubes  or  Petri  dishes  at  a  time.  For  this 
purpose  the  jar  invented  by  Novy  or  the  apparatus  of  Botkin  can 
be  used. 

The  Novy  jar  receives  as  many  inoculated  tubes  as  it  will  contain 
and  has  its  stopper  so  replaced  that  the  openings  in  the  neck  and 
stopper  correspond.  Hydrogen  gas  is  passed  through  until  the  air 
is  displaced.  This  usually  takes  several  hours,  as  the  cotton  stop- 
pers retain  the  air  in  the  test-tubes  and  prevent  rapid  diffusion. 
When  the  air  is  all  displaced,  the  stopper  is  turned  so  that  the  tubes 
are  closed.  If  it  be  desired  to 
expedite  matters  a  pump  can  be 
used  to  withdraw  the  air,  after 
which  the  hydrogen  is  permitted 
to  enter. 

Botkin's  apparatus  is  intended 
for  cultures  in  Petri  dishes.  It 
consists  of  three  parts — a  deep 
dish  of  glass  (7>),  a  stand  to  sup- 
port the  Petri  dishes  to  be  ex- 
posed (c),  and  a  bell-glass  (a)  to 
cover  the  stand  and  fit  inside  of 
the  dish.  The  prepared  dishes 
are  stood  uncovered  in  the  rack, 
which  is  then  placed  in  the  dish 
forming  the  bottom  of  the  appa- 
ratus, and  into  which  liquid  par- 
affin is  poured  to  a  depth  of  about  ,.,. 

.      i  rp,,      i    n     i  .       tig.  65. — Botkin's  apparatus  for  mak- 

2  inches.     The  bell-glass  cover  is  ing  anaerobic  cultures. 

now  stood  in  place  and  hydrogen 

gas  is  conducted  through  previously  arranged  rubber  tubes  (d,  e)'. 

As  soon  as  the  air  is  displaced  through  tube  d,  both  tubes  are 

withdrawn.     It  is  well  to  place  one  Petri  dish  containing  alkaline 

pyrogallic  acid  in  the  rack  to  absorb  any  oxygen  not  successfully 

displaced. 

3.  The  Absorption  of  the  Atmospheric  Oxygen. — This  method 
was  first  suggested  by  Buchner,  whose  idea  was  to  absorb  the  atmos- 
pheric oxygen  by  alkaline  pyrogallic  acid  and  permit  the  bacteria 
to  develop  in  the  indifferent  nitrogen.  Various  methods  have  been 
suggested  for  achieving  this  end,  Buchner 's  own  method  consisting 
in  the  use  of  two  tubes,  a  small  one  to  contain  the  culture  and  a 
larger  one  to  contain  the  absorbing  fluid.  A  fresh  solution  of  pyro- 
gallic acid  and  sodium  "hydroxid  were  poured  into  the  large  tube, 
the  smaller  tube  placed  within  it,  upon  some  appropriate  support, 
and  the  whole  tightly  corked. 

Nichols  and  Schmitter,  *  at  the  suggestion  of  Carroll,  have  modified 
*  "Jour,  of  Medical  Research,"  1906,  xv,  p.  113. 


218 


The  Cultivation  of  Anaerobic  Organisms 


the  method  by  connecting  the  tube  containing  the  inoculated  cul- 
ture medium  with  a  U-shaped  tube,  to  the  other  end  of  which  is 
attached  a  tube  to  contain  the  pyrogallic  acid  solution.  The  ap- 
paratus will  at  once  be  understood  by  a  glance  at  the  cut.  The 
mode  of  employing  it  is  as  follows:  " After  inoculating  the  cul- 
ture-tube the  plug  is  pushed  in  a  little  below  the  lips  of  the  tube; 
the  ends  of  the  U  tube  and  the  test-tubes  are  coated  externally  with 
vaselin,  the  rubber  tubes  are  adjusted  on  the  U  tube  and  a  connec- 
tion made  with  the  culture-tube  so  that 
the  glass  ends  meet.  One  or  two  grams 
of  pyrogallic  acid  are  put  in  the  empty 
test-tube,  and  packed  down  with  a  little 
filter-paper  over  it;  ten  or  twenty  cubic 
centimeters,  respectively,  of  a  10  per 
cent,  solution  of  sodium  hydroxide  are 
then  poured  into  the  tube  and  the  second 
connection  made  before  the  acid  and 
alkali  react  to  any  extent." 

Wright  has  suggested  that  the  cotton 
stopper    of    the    ordinary    culture-tube 
have  its  projecting  part  cut  off  and  the 
plug  itself  pushed  down  the  tube  for  a 
short  distance.     Some  alkaline  pyrogallic 
acid  solution  is  poured  upon  the  cotton, 
to  saturate  it,  and  the  tube  tightly  corked. 
Zinsser*  has  recommended  the  follow- 
ing method  as  satisfactory  for  use  with 
Petri  dishes.     The  dishes  selected  should 
be   rather  deeper  than  ordinary.     They 
are  sterilized  and  inoculated  in  the  ordi- 
nary manner  and   then   inverted.     The 
Fig.     66.-SPirillum    ru-     dish  is  cautiously  raised,  and  some  pyro- 
brum.      Glucose  agar  slant     gallic  acid  carefully  poured  into  the  lid 
culture  of  five  days.    Abun-     and  the  dish  gently  dropped  into  place 

again.     The    alkaline    solution    is    then 
poured  into  the  crevice  between  the  edges 


photographer.) 
and  Schmitter.) 


dant  production  of  pigment 
on  the  surface.  (The  U  tube 
was  soiled  by  the  reducing 
fluid  during  handling  by  ^the  of  the  dish  and  the  lid?  and  the  remain. 

der  of  the  space  filled  with  melted  albo- 
lene.  When  these  dishes  are  carefully 
stood  away,  the  alkaline  pyrogallic  acid  absorbs  all  of  the  con- 
tained oxygen  and  the  anaerobic  cultures  develop  quite  well.  The 
growing  colonies  can  be  examined  as  often  as  may  be  necessary 
through  the  bottom'  of  the  dishes,  which  must,  of  course,  always  be 
kept  in  the  inverted  position. 

4.  Reduction  of  Oxygen. — Pasteur  and,  later,  Roux  have  recom- 
mended "the  cultivation  of  anaerobic  bacteria  in  association  with 
*  "Journal  of  Experimental  Medicine,"  1906,  vm,  542. 


Exclusion  of  Atmospheric  Oxygen 


219 


aerobic  bacteria  by  which  the  oxygen  was  to  be  absorbed.  This 
method  is  too  crude  to  be  employed  at  the  present  time,  as  it  destroys 
the  essential  characteristics  of  the  cultures  by  mixing  the  products 
of  the  bacteria. 

Chemic  reduction  of  the  oxygen  has  been  attempted  by  the  addi- 
tion of  2  per  cent,  of  glucose,  as  suggested  by  Liborius,  0.3-0.5  per 
cent,  of  sodium  formate,  as  suggested  by  Kitasato  and  Weil,  o.i 
per  cent,  of  sodium  sulphate,  suggested  by  the  same  authors,  and 
various  other  chemicals.  None  of  these  additions  has  been  suffi- 
ciently successful  to  merit  continued  favor,  and  at  the  present  time 
this  method  is  not  employed. 

5.  Exclusion  of  Atmospheric  Oxygen  by  Means  of  Various 
Physical  Principles  and  Mechanical  Devices. — This  has  appealed 
to  the  ingenuity  of  many  experimenters,  and  many  means  of  accom- 
plishing it  have  been  tried  with  success. 


Fig.  67. — Buchner's  method  of  mak- 
ing anaerobic  cultures. 


Fig.  68. — Hesse's  method  of  making 
anaerobic  cultures. 


The  most  simple  plan  is  that  of  Hesse,  who  made  a  deep  puncture 
in  recently  boiled  and  rapidly  cooled  gelatin  or  agar-agar,  then  cov- 
ered the  surface  of  the  medium  with  sterile  oil.  The  so-called 
" shake  culture"  is  another  very  simple  method,  suggested  by 
Liborius  and  Hesse.  The  medium  to  be  inoculated,  contained  in  a 
well-filled  tube  or  flask,  is  boiled  to  displace  the  contained  air,  cooled 
so  as  no  longer  to  endanger  the  introduced  bacteria,  then  inoculated, 
the  inoculated  bacteria  being  distributed  by  gently  shaking.  On 
cooling,  the  medium  "sets,"  the  organisms  below  the  surface  remain- 
ing under  anaerobic  conditions. 

Kitasato  first  used  paraffin  as  a  covering  for  the  inoculated  medium, 


220 


The  Cultivation  of  Anaerobic  Organisms 


his  recommendation  having  recently  been  revived  by  Park  and  made 
successful  for  the  cultivation  of  the  tetanus  bacillus.  The  paraffin 
floats  upon  the  surface  of  the  medium,  melts  during  sterilization, 
but  does  not  mix  with  it,  and  "sets"  when  cool.  The  inoculation 
is  to  be  made  while  the  culture  medium  is  warm,  after  boiling  and 
before  the  paraffin  sets. 

Koch  studied  the  colonies  of  anaerobic  organisms  by  cultivating 
them  upon  a  film  of  gelatin  covered  by  a 
thin  sheet  of  sterilized  mica,  by  which  the 
air  was  excluded. 

Salamonsen  has  made  use  of  a  pipet  for 
making  anaerobic  cultures.  It  is  made 
of  a  glass  tube  a  few  millimeters  in  diam- 
eter, drawn  out  to  a  point  at  each  end. 
The  inoculated  gelatin  or  agar-agar  is 
drawn  in  while  liquefied  and  the  ends 
sealed.  The  tube,  of  course,  contains  no 
air,  and  perfect  anaerobiosis  results. 

Theobald  Smith  has  found  the  fer- 
mentation-tube and  various  modifications 
of  it  excellently  well  adapted  to  the 
growth  of  anaerobes,  which,  of  course, 
grow  only  in  the  closed  limb. 

Hens'  eggs  have  been  used  for  anaerobic 
cultures,  and  in  them  the  tetanus  bacillus 
grows  remarkably  well.  Conditions  of 
anaerobiosis  are,  however,  not  perfect,  as 
can  be  shown  by  the  behavior  of  the  egg 
itself.  If  oxygen  be  completely  shut  out 
by  oiling  or  varnishing  the  shell,  a  fertile 
egg  will  not  develop. 

A  quite  satisfactory  and  simple  device 
for  routine  work  with  anaerobic  organisms 
has  been  invented  by  Wright.*    The  es- 
sential feature  consists  of  a  pipet,  D,  with 
Figs    69,    70.— Wright's    a  rubber  tube,  E,  at  the  end,  and  one  in- 
method  of  making  anaero-  .  ,     ,  ,  i  i_       ,    i        /-• 

bic  cultures  in  fluid  media  terruption  connected  by  a  rubber  tube,  C. 
(Mallory  and  Wright).  The  device  will  be  made  clear  at  once  by 

a  glance  at  the  accompanying  illustration. 

The  method  of  employment  is  very  simple.  An  ordinary  tube  of 
bouillon  or  other  fluid  culture- media  receives  the  pipet,  the  whole 
being  sterilized,  the  cotton  plug  in  place.  The  bouillon  being  in- 
oculated with  the  culture  or  secretion  to  be  studied  is  drawn  up 
in  the  bulb  of  the  pipet,  A,  by  suction,  until  it  passes  the  rubber  inter- 
ruption, C.  By  forcing  the  upper  end  of  the  pipet  downward  in  the 


*  "Jour.  Boston  Soc.  of  Med.  Sci.,"  Jan.,  1900. 


Exclusion  of  Atmospheric  Oxygen  221 

test-tube,  a  kink  is  given  each  rubber  tube  and  the  fluid  contained 
in  the  bulbous  part  of  the  pipet  becomes  hermetically  sealed. 

In  all  cases  where  the  presence  of  suspected  micro-organisms  is  to 
be  demonstrated,  it  is  necessary  to  make  both  aerobic  and  anaerobic 
cultures.  For  routine  work  of  this  kind,  this  method  of  Wright  is 
probably  the  most  convenient  yet  suggested. 


CHAPTER  X 
EXPERIMENTATION  UPON  ANIMALS 

THE  principal  objects  of  medical  bacteriology  are  to  discover 
the  cause,  explain  the  symptoms,  and  bring  about  the  cure  and 
future  prevention  of  disease.  We  cannot  hope  to  achieve  these  ob- 
jects without  experimentation  upon  animals,  in  whose  bodies  the 
effects  of  bacteria  and  their  products  can  be  studied. 

No  one  should  more  heartily  condemn  wanton  cruelty  to  animals 
than  the  physician.  Indeed,  it  is  hard  to  imagine  men,  so  much  of 
whose  life  is  spent  in  relieving  pain,  and  who  know  so  much  about 
pain,  being  guilty  of  the  butchery  and  torture  accredited  to  them  by 
a  few  of  the  laity,  whose  eyes,  but  not  whose  brains,  have  looked  over 
the  pages  of  text-books  of  physiology,  and  whose  "  philanthropy  has 
thereby  been  transformed  to  zoolatry." 


Fig.  71. — r,  Roux's  bacteriologic  syringe;  2,  Koch's  syringe;  3,  Meyer's 
bacteriologic  syringe.  Such  syringes,  because  of  their  complexity  and  the 
destructible  packings,  give  very  unsatisfactory  service  and  are  no  longer  em- 
ployed. 

It  is  largely  through  experimentation  upon  animals  that  we  have 
attained  our  knowledge  of  physiology,  most  of  our  important  knowl- 
edge of  therapeutics,  and  most  of  our  knowledge  of  the  infectious 
diseases.  Without  its  aid  we  would  still  be  without  one  of  the  great- 
est achievements  of  medicine,  the  "blood  serum  therapy." 

Experiments  upon  animals,  therefore,  must  be  made,  and,  as  the 
lower  animals  differ  in  their  susceptibility  to  diseases,  large  numbers 
and  different  kinds  of  animals  must  be  employed. 

The  bacteriologic  methods  are  fortunately  not  cruel,  the  principal 
modes  of  introducing  bacteria  into  the  body  being  by  subcutaneous, 
intraperitoneal,  and  intravenous  injection. 

Hypodermic  syringes,  expressly  designed  for  bacteriologic  work 

222 


Animal  Inoculations 


223 


are  shown  in  the  illustration.     Those  of  Meyer  and  Roux  resemble 
ordinary  hypodermic  syringes;  that  of  Koch  is  supposed  to  possess 


Fig.  72.— Altmann  syringes  for  bacteriologic  and  hematologic  work.     These  are 
capable  of  sterilization  without  injury  and  are  thoroughly  satisfactory. 


Fig.  73- — Method  of  making  an  intravenous  injection  into  a  rabbit.     Observe 
that  the  needle  enters  the  posterior  vein  from  the  hairy  surface. 

the  decided  advantage  of  not  having  a  piston  to  come  into  contact 
with  the  fluid  to  be  injected.  This  is,  however,  really  disadvanta- 
geous, inasmuch  as  the  cushion  of  compressed  air  that  drives  out  the 


224  Experimentation  upon  Animals 

contents  is  elastic,  and  unless  carefully  watched  will  follow  the  injec- 
tion into  the  body  of  the  animal.  In  making  subcutaneous  injec- 
tions there  is  no  disadvantage  or  danger  from  the  entrance  of  air, 
but  in  intravenous  injections  it  is  extremely  dangerous. 

Syringes  with  metal  or  glass  pistons  like  those  shown  are  to  be 
preferred.  All  syringes  should  be  disinfected  by  boiling  thoroughly, 
before  and  after  using.  Syringes  with  packings  to  tighten  the  pistons 
cannot  be  boiled  with  impunity,  as  it  soon  ruins  them,  and  new  pack- 
ings may  be  difficult  to  obtain  or  fit.  Syringes  of  such  design  should 
be  avoided. 

The  intravenous  injection  is  easy  to  achieve  in  a  large  animal,  like 
a  horse,  but  is  very  difficult  in  animals  smaller  than  a  rabbit.  Such 
injections,  when  given  to  rabbits,  are  usually  made  into  the  ear- 
veins,  which  are  most  conspicuous  and  accessible.  A  peculiar  and 
important  fact  to  remember  is  that  the  less  conspicuous  posterior 
vein  of  the  ear  is  much  better  adapted  to  the  purpose  than  the  an- 
terior. The  introduction  of  the  needle  should  be  made  from  the 
hairy  external  surface  of  the  ear  where  the  vein  is  immediately  beneath 
the  skin. 

If  the  ear  be  manipulated  for  a  moment  or  two  before  the  injection, 
vasomotor  dilatation  occurs  and  the  blood-vessels  become  larger  and 
more  conspicuous.  The  vein  should  be  compressed  at  the  root  of  the 
ear  until  the  needle  is  introduced,  and  the  injection  made  as  near  the 
root  as  possible.  The  fluid  should  be  injected  slowly. 

By  using  very  fine  needles,  similar  injections  may  be  made  into  the 
ear  veins  of  guinea-pigs.  By  dipping  the  tails  of  rats  and  even  mice 
into  warm  water  so  as  to  cause  dilatation  of  the  caudal  veins,  it  may 
be  possible  to  effect  intravenous  injections  of  such  animals.  Kolmer 
suggests  that  the  tails  be  vigorously  rubbed  with  xylol  or  alcohol, 
and  the  epidermal  cells  softened  and  scraped  off  so  as  to  expose  the 
veins  better.  As  the  first  attempt  to  get  the  needle  into  the  caudal 
vein  may  fail,  and  new  attempts  be  required,  it  is  well  to  begin  at  a 
point  not  too  near  the  body. 

Bacteria  can  be  introduced  into  the  lymphatics  only  by  injecting 
liquid  cultures  into  some  organ  with  comparatively  few  blood-vessels 
and  large  numbers  of  lymphatics.  The  testicle  is  best  adapted  to 
this  purpose,  the  needle  being  introduced  deeply  into  the  organ. 

Sometimes  subcutaneous  inoculations  are  made  by  introducing  the 
platinum  wire  through  a  small  opening  made  in  the  skin  by  a  snip  of 
the  scissors.  By  this  means  solid  cultures  from  agar-agar,  etc.,  can  be 
introduced. 

Intra-abdominal  and  intrapleural  injections  are  sometimes  made, 
and  in  cases  where  it  becomes  necessary  to  determine  the  presence 
or  absence  of  the  bacilli  of  tuberculosis  or  glanders  in  fragments  of 
tissue  it  may  be  necessary  to  introduce  small  pieces  of  the  suspected 
tissue  under  the  skin.  To  do  this  the  hair  is  closely  cut  over  the 
point  of  election,  which  is  generally  on  the  abdomen  near  the  groin, 


Animal  Inoculations  225 

the  skin  picked  up  with  forceps,  a  snip  made  through  it,  and  the  points 
of  the  scissors  introduced  for  an  inch  or  so  and  then  separated.     By 


Fig.  74. — Latapie's  animal  holder  for  rabbits,  guinea-pigs,  and  other 
small  animals.  This  form  of  holder  is  in  general  use  at  the  Institute  Pasteur  in 
Paris. 

this  manoeuver  a  subcutaneous  pocket  is  formed,  into  which  the 
tissue  is  easily  forced.  The  opening  should  not  be  large  enough  to 
require  subsequent  stitching. 


Fig.  75. — Guinea-pig  confined  in  the  holder. 

When  tissue  fragments  or  collodion  capsules  are  to  be  introduced 
into  the  abdominal  cavity,  the  animal  should  be  anesthetized  and 


Fig.  76. — Mouse-holder. 

a  formal   laparotomy   done,  the   wound  being  carefully  stitched 
together. 


226  Experimentation  upon  Animals 

When,  in  studying  Pfeitfer's  phenomenon  and  similar  conditions, 
•it  is  desirable  occasionally  to  withdraw  drops  of  fluid  from  the  ab- 
dominal cavity,  a  small  opening  can  be  burned  through  with  a  blunt 
needle.  This  does  not  heal  readily,  and  through  it,  from  time  to 
time,  a  capillary  pipet  can  be  introduced  and  the  fluids  withdrawn. 

Small  animals,  such  as  rabbits  and  guinea-pigs,  can  be  held  in  the 
hand,  as  a  rule.  Guinea-pig  and  rabbit-holders  of  various  forms 
can  be  obtained  from  dealers  in  laboratory  supplies.  The  best  of 
these  is  undoubtedly  that  of  Latapie,  shown  in  the  accompanying 
illustration.  Dogs,  cats,  sheep,  and  goats  can 
be  tied  and  held  in  troughs.  A  convenient 
form  of  mouse-holder,  invented  by  Kitasato, 
is  shown  in  the  figure. 

In  all  these  experiments  one  must  remember 
that  the  amount  of  material  introduced  into 
the  animal  must  be  in  proportion  to  its  size, 
and  that  injection  experiments  upon  mice  are 
usually  so  crude  and  destructive  as  to  warrant 
the  comparison  drawn  by  Frankel,  that  the 
injection  of  a  few  minims  of  liquid  into  the 
pleural  cavity  of  a  mouse  is  "much  the  same 
as  if  one  would  inject  through  a  fire-hose  three 
or  four  quarts  of  some  liquid  into  the  respira- 
tory organs  of  a  man." 

Method  of  Securing  Blood  from  Animals. — 
For  many  experimental  purposes  it  becomes 
necessary  to  secure  blood  in  larger  or  smaller 
quantities  from  animals.  For  horses,  cattle, 
calves,  goats,  sheep,  large  dogs,  etc.,  this  is  a 
simple  matter,  all  that .  is  necessary  being  to 
restrain  the  animal,  make  a  minute  incision  in 

b7loodTfrom  the  the  ^  OVer  the  W1"  veuin>  whi*  *  easily 
carotid  artery  of  a  found  by  compressing  it  at  the  root  of  the  neck 
rabbit  or  guinea-pig.  and  noting  where  the  vessel  expands,  and  in- 
troducing a  canula  when  the  vein  is  well  dis- 
tended. The  trocar  being  withdrawn,  the  blood  at  once  flows.  A 
sterile  tube  is  slipped  over  the  canula  and  the  blood  conducted  into 
a  sterile  bottle  or  flask. 

For  rabbits  and  guinea-pigs  the  technic  is  rather  more  difficult 
because  of  the  smaller  size  of  the  vessels.  Drops  and  small  quanti- 
ties of  blood  may  be  secured  by  opening  one  of  the  ear  veins,  but 
when  any  quantity  of  blood  is  required,  the  neatest  operation  is 
done  by  tapping  the  common  carotid  artery  by  the  method  employed 
at  the  Pasteur  Institute  at  Paris. 

The  animal  is  restrained  in  a  Latapie  holder,  with  the  neck  ex- 
tended." Anesthesia  can  be  used,  but  must  be  employed  with  great 
care.  The  hair  on  the  front  of  the  neck  is  clipped  and  the  neck 


Securing  Blood  from  Animals 


227 


shaved,  or,  as  is  easier,  the  hair  is  pulled  out,  leaving  a  clean  surface 
an  inch  square.  The  skin  is  then  washed  with  a  disinfecting  solu- 
tion, an  incision  one  and  a  half  inches  long  made  through  the  skin 
and  superficial  fascia  in  the  middle  line  of  the  neck,  the  tissues  care- 
fully separated,  the  deep  fascia  cautiously  opened,  the  tissues  sepa- 
rated with  the  point  of  the  forceps  and  a  grooved  director,  the 
sheath  of  the  vessels  opened,  and  the  artery  completely  separated 
from  its  surrounding  tissues  for  a  distance  of  at  least  an  inch.  A 
ligature  is  now  tightly  tied  about  the  artery  at  the  distal  end  of  ex- 
posure, and  a  ligature  placed  in  position  and  loosely  looped  ready  to 
tie  about  the  proximal  end.  A  tube  with  a  sharp  lateral  tubulature, 
as  is  shown  in  the  illustration,  is  now  made  ready  by  breaking  off 


Fig.  78. — Showing  the  method  of  taking  blood  from  the  carotid  artery 

of  a  rabbit. 

the  closed  tip,  the  moistened  forefinger  of  the  operator  is  placed 
beneath  the  artery,  and  the  sharp  tube  inserted  (point  toward  the 
heart)  into  the  artery,  through  whose  walls  it  cuts  its  way  easily. 
The  moment  the  vessel  is  entered  the  blood-pressure  drives  the  blood 
into  the  tube  so  that  20  cc.  maybe  collected  in  about  as  many  seconds. 
An  assistant  now  ties  the  artery  at  its  proximal  end,  the  tube  is  with- 
drawn, holding  it  so  that  the  blood  does  not  escape,  and  the  end 
sealed  in  a  flame.  The  ends  of  the  ligatures  are  now  cut  short  and 
the  external  wound  stitched.  The  wound  usually  heals  at  once,  and 
if  subsequent  study  of  the  blood  is  required,  the  other  carotid  and 
the  femorals  can  be  similarly  employed  for  obtaining  it. 

Small  quantities  of  blood  (drops)  can  be  secured  from  mice  and 
rats  by  cutting  off  the  tip  of  the  tail,  but  to  secure  a  large  quantity 


228  Experimentation  upon  Animals 

is  difficult.  One  method  that  has  been  recommended  is  to  tie  the 
animal  to  a  tray  or  board,  on  its  back,  anesthetize  it,  and,  just  before 
it  dies,  quickly  open  the  thoracic  cavity,  and  cut  through  the  heart 
with  scissors.  The  animal  at  once  dies,  the  blood  pouring  out  into 
the  pleural  cavities.  After  coagulation  the  serum  can  be  secured 
by  carefully  pipetting  it  from  the  cavities. 

Post-mortems. — Observation  of  experiment  animals  by  no  means 
ceases  with  their  death.  Indeed,  he  cannot  be  a  bacteriologist  who 
is  not  already  a  good  pathologist  and  expert  in  the  recognition  of 
diseased  organs. 

When  an  autopsy  is  to  be  made  upon  a  small  animal,  it  is  best 
to  wash  it  for  a  few  moments  in  a  disinfecting  solution,  to  kill  the 
germs  present  upon  the  hair  and  skin,  as  well  as  to  moisten  the  hair, 
which  can  then  be  much  more  easily  kept  out  of  the  incision. 

Small  animals  can  be  tacked  to  a  board  or  tied,  by  cords  fastened 
to  the  legs,  to  hooks  soldered  to  the  corners  of  an  easily  disinfected 
tray.  The  dissection  should  be  made  with  sterile  instruments. 
When  a  culture  is  to  be  made  from  the  interior  of  an  organ,  its  surface 
should  first  be  seared  with  a  hot  iron,  a  puncture  made  into  it  with  a 
sterile  knife,  and  the  culture  made  by  introducing  a  platinum  wire. 

If  the  bacteriologic  examination  cannot  be  made  at  once,  the  or- 
gans to  be  studied  should  be  removed  with  aseptic  precautions, 
wrapped  in  a  sterile  towel  or  a  towel  wet  with  a  disinfecting  solution, 
and  carried  to  the  laboratory,  where  the  surface  is  seared  and  the 
necessary  incisions  made  with  sterile  instruments. 

Fragments  intended  for  subsequent  microscopic  examination 
should  be  cut  into  small  cubes  (of  i  cc.)  and  fixed  in  Zenker's  fluid 
or  absolute  alcohol. 

Collodion  capsules  are  quite  frequently  employed  for  the  purpose 
of  cultivating  bacteria  in  a  confined  position  in  the  body  of  an  animal, 
where  they  can  freely  receive  and  utilize  the  body-juices  without 
being  subjected  to  the  action  of  the  phagocytes.  In  such  capsules 
the  bacteria  usually  grow  plentifully,  and  not  rarely  their  virulence 
is  increased. 

The  capsules  can  be  made  of  any  size,  though  they  are  probably 
most  easily  handled  when  of  about  5-10  cc.  capacity.  The  size  is 
always  an  objection,  because  of  the  disturbance  occasioned  when 
they  are  introduced  into  the  abdominal  cavity. 

The  capsules  are  made  by  carefully  coating  the  outside  of  the 
lower  part  of  a  test-tube  with  collodion  until  a  sufficiently  thick, 
homogeneous  layer  is  formed.  During  the  coating  process  the  tube 
must  be  twirled  alternately  within  and  without  the  collodion,  so 
that  it- is  equally  distributed  upon  its  surface.  When  the  desired 
thickness  is  attained,  and  the  collodion  is  sufficiently  firm,  the  tube 
is  plunged  under  water  and  the  hardening  process  checked. 

A  cut  is  next  made  around  the  upper  edge  of  the  collodion  film, 
and  it  is  removed  by  carefully  turning  it  inside  out.  In  this  manner 


Collodion  capsules 


229 


an  exact  mold  of  the  tube  is  formed.  If  a  small  opening  be  made 
at  the  end  of  the  tube  over  which  the  sac  is  molded,  and  the  tube 
filled  with  water  after  being  properly  coated  with  collodion,  a  small 
amount  of  pressure,  applied  by  blowing  gently  into  the  tube,  will 
force  the  water  between  the  collodion  and  glass  and  so  detach  it 
without  inversion.  A  test-tube  of  the  same  size  is  next  constricted 
to  a  degree  that  will  not  interfere  with  the  future  introduction  of 
cukure-media  in  a  fine  pipet  or  inoculation  with  a  platinum  loop, 
and  that  will  permit  of  ready  sealing  in  a  flame  when  necessary; 
the  rounded  end  is  cut  off,  and  the  edges  are  smoothed  in  a  flame. 
The  upper  open  end  of  the  collodion  bag  is 
carefully  fitted  over  the  end  of  the  tube,  shrunk 
on  by  a  gentle  heating,  and  cemented  fast 
with  a  little  fresh  collodion  applied  to  the  line 
of  union.  rNovy  recommends  that  a  thread 
of  silk  be  wound  around  the  point  of  union,  to 
hold  the  collodion  in  place  and  to  aid  in  han- 
dling the  finished  sac.  The  sac  is  next  filled 
with  distilled  water  up  to  the  thread,  the  tube 
is  plugged  with  cotton,  and  the  whole  placed 
in  a  larger  test-tube  containing  distilled  water, 
the  cotton  plug  being  packed  tightly  around 
the  smaller  tube,  so  that  the  collodion  sac  does 
not  reach  the  bottom  of  the  large  tube,  but 
hangs  suspended  in  the  water  it  contains.  The 
whole  is  now  carefully  sterilized  by  steam. 

When  ready  for  use,  a  tube  of  bouillon  is  in-  Jjd.Sto'Si  &£=  ^ 
oculated  with  the  culture  intended  to  be  placed 
in  the  animal,  the  water  in  the  capsule  is  pipetted  out  and  replaced 
by  the  inoculated  bouillon  carefully  introduced  with  a  pipet,  the  con- 
stricted portion  is  sealed  in  a  flame,  and  the  capsule  picked  up  with 
forceps  is  introduced  into  the  peritoneal  cavity  by  an  aseptic 
operation. 

The  collodion  capsules  may  be  made  of  any  size.  Those  for  rabbit 
experiments  should  be  of  about  10  cc.  capacity,  those  for  guinea-pig 
experiments  about  5  cc.  By  coating  large  glass  tubes  they  can  be 
made  of  500  cc.  capacity,  the  large  bags  being  useful  for  chemic 
dialysis. 


Fig.  7  9  .—Prepara- 
tion of  collodion  sacs: 
a.  Test-tube  constric- 


CHAPTER  XI 
THE  IDENTIFICATION  OF  SPECIES 

THE  most  difficult  thing  in  bacteriology  is  the  identification  of 
the  species  of  bacteria  that  come  under  observation. 

A  few  micro-organisms  are  characteristic  in  morphology  and  in 
their  chemic  and  other  products,  and  present  no  difficulty.  Thus, 
the  tubercle  bacillus  is  characteristic  in  its  reaction  to  the  anilin 
dyes,  and  can  usually  be  recognized  by  this  peculiarity.  Some,  as 
Bacillus  mycoides,  have  characteristic  agar-agar  growths.  The  red 
color  of  Bacillus  prodigiosus  and  the  blue  of  Bacillus  janthinus  speak 
almost  positively  for  them.  The  potato  cultures  of  Bacillus  mesen- 
tericus  fuscus  and  vulgatus  are  usually  sufficient  to  enable  us  to 
recognize  them.  Unfortunately,  however,  there  are  several  hun- 
dreds of  described  species  that  lack  any  one  distinct  characteristic 
that  may  be  used  for  differential  purposes,  and  require  that  for  their 
recognition  we  shall  well-nigh  exhaust  the  bacteriologic  technic. 

Tables  for  the  purpose  have  been  compiled  by  Eisenberg,  Migula, 
Lehman  and  Neumann,  Chester,  and  others,  and  are  indispensable 
to  the  worker.  The  most  useful  are  probably  the  "Atlas  and  Grun- 
driss  der  Bakteriologie  und  Lehrbuch  der  speziellen  bakteriologischen 
Diagnostik,"  by  Lehmann  and  Neumann,*  and  the  "  Manual  of 
Determinative  Bacteriology,"  by  F.  D.  Chester  (1901),  from  which, 
through  the  courtesy  of  the  author  and  publisher,  the  following 
synopsis  of  groups  is  taken.  Unfortunately,  in  tabulating  bacteria 
we  constantly  meet  species  described  so  insufficiently  as  to  make  it 
impossible  to  properly  classify  and  tabulate  them. 

The  only  way  to  determine  a  species  is  to  study  it  thoroughly, 
step  by  step,  and  compare  it  with  the  description  and  tables.  In 
this  regard  the  differentiation  of  bacteria  resembles  the  determina- 
tion of  the  higher  plants  with  the  aid  of  a  botanic  key,  or  the  qualita- 
tive analysis  for  the  detection  of  unknown  chemic  compounds. 
Such  a  key  for  specific  bacterial  differentiation  is  really  indispensa- 
ble, even  though  it  be  imperfect,  and  every  student  engaged  in  re- 
search work  should  have  one.  As  Chester  says:  "probably  nine- 
tenths  of  the  forms  of  bacteria  already  described  might  as  well  be 
forgotten  or  given  a  respectful  burial.  This  will  then  leave  com- 
paratively few  well-defined  species  to  form  the  nuclei  of  groups  in 
one  or  another  of  which  we  shall  be  able  to  place  all  new  and  suffi- 
ciently described  forms."  "That  typical  forms  or  species  of  bac- 
teria do  exist,  no  one  can  deny.  These  typical  forms  furthermore 

*  J.  F.  Lehmann,  Miinchen,  1907. 
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Chester's  Synopsis  of  Groups  of  Bacteria  231 

present  certain  definite  morphologic,  biologic,  cultural,  and  perhaps 
pathogenic  characters  which  establish  the  types  independently  of 
minor  variations. 

"The  most  marked  of  these  types  we  select  to  become  the  centers 
of  groups,  around  which  are  gathered  all  related  species  or  varieties." 
"  The  division  of  the  bacteria  into  groups,  so  far  as  grouping  was  pos- 
sible, is  outlined  in  the  following  tables:" 

A  PROPOSED  SYNOPSIS  OF  GROUPS  OF  BACTERIA 

BACTERIUM 

I.  Without  endospores. 

A.  Aerobic  and  facultative  anaerobic. 

a.  Gelatin  not  liquefied. 

*  Decolorized  by  Gram's  method. 

t  Obligate  aerobic.     ACETIC  FERMENT  GROUP. 
tf  Aerobic  and  facultative  anaerobic. 
Gas  generated  in  glucose  bouillon. 

Gas    generated   in   lactose    bouillon.     BACT.    AEROGENES 

GROUP. 

Little   or   no   gas   generated  in  lactose  bouillon.    FRIED- 
LANDER  GROUP. 

No  gas  generated  in  glucose  bouillon. 
Milk  coagulated.     FOWL  CHOLERA  GROUP. 
Milk  not  coagulated.     SWINE  PLAGUE  GROUP. 
**  Stained  by  Gram's  method. 

f  Gas  generated  in  glucose  bouillon.     LACTIC  FERMENT  GROUP. 

b.  Gelatin  liquefied. 

*  Colonies  on  gelatin  ameboid  or  proteus-like.     BACT.  RADIATUM 

GROUP. 
**  Colonies  on  gelatin  round,  not  ameboid.     BACT.  AMBIGUUM  GROUP. 

II.  Produce  endospores. 

1.  No  growth  at  room  temperature,  or  below   22°-25°C.     THERMOPHILIC 

GROUP. 

2.  Grow  at  room  temperatures. 

a.  Gelatin  liquefied.     ANTHRAX  GROUP. 

b.  Gelatin  not  liquefied.     BACT.  F^ECALIS  GROUP. 

BACILLUS 
I.  Without  endospores. 

A.  Aerobic  and  facultative  anaerobic. 

a.  Gelatin  colonies  roundish,  not  distinctly  ameboid. 
*  Gelatin  not  liquefied. 

f  Decolorized  by  Gram's  method. 
Gas  generated  in  glucose  bouillon. 
Milk  coagulated.     COLON  GROUP.  - 
Milk  not  coagulated.     HOG  CHOLERA  GROUP. 
No  gas  generated  in  glucose  bouillon.     TYPHOID   GROUP. 
ft  Stained  by  Gram's  method.     B.  MURIPESTIFER  GROUP. 
**  Gelatin  liquefied. 

t  Gas  generated  in  glucose  bouillon.     B.  CLOACAE  GROUP. 
tfNo  gas  generated  in  glucose  bouillon.     Include  a  large  number 

of  bacteria  not  sufficiently  described  to  arrange  in  groups. 
b.  Gelatin  colonies  ameboid,  cochleate,  or  otherwise  irregular. 
*  Gelatin  liquefied.     PROTEUS  VULGARIS  GROUP.  » 
**  Gelatin  not  liquefied.     B.  ZOPFI  GROUP. 
II.  Produce  endospores. 

A.  Aerobic  and  facultative  anaerobic, 
i.  Rods  not  swollen  at  sporulation. 
a.  Gelatin  liquefied. 


232  The  Identification  of  Species 

*  Liquefaction  of  the  gelatin  takes  place  slowly.     Ferment 
urea,  with  strong  production  of  ammonia.     URO-BACILLUS 
GROUP  OF  MIQUEL. 
**  Gelatin  liquefied  rather  quickly. 

t  Potato  cultures  rugose.     POTATO  BACILLUS  GROUP. 
tf  Potato  cultures  not  distinctly  rugose.     B.  SUBTILIS  GROUP. 
b.  Gelatin  not  liquefied.     B.  SOLI  GROUP. 

2.  Rods  spindle-shaped  at  sporulation.     B.  LICHENIFORMIS  GROUP. 

3.  Rods  clavate  at  sporulation.     B.  SUBLANATUS  GROUP. 
B.  Obligate  anaerobic. 

1.  Rods  not  swollen  at  sporulation.     MALIGNANT  EDEMA  GROUP. 

2.  Rods  spindle-shaped  at  sporulation.     CLOSTRIDIUM  GROUP. 

3.  Rods  clavate-capitate  at  sporulation.     TETANUS  GROUP. 

PSEUDOMONAS  (Migula) 

I.  Cells  colorless,  without  a  red-colored  plasma  and  without  sulphur  granules. 

A.  Grow  in  ordinary  culture-media. 

i.  Without  endospores. 

a.  Aerobic  and  facultative  anaerobic. 

*  Without  pigment. 

t  Gelatin  not  liquefied. 

Gas    generated    in    glucose    bouillon.      Ps.    MONADIFORMIS 

GROUP. 

No  gas  generated  in  glucose  bouillon.     Ps.  AMBIGUA  GROUP. 
ft  Gelatin  liquefied. 

Gas  generated  in  glucose  bouillon.     Ps.  COADUNATA  GROUP. 
No  gas  generated  in  glucose  bouillon.     Ps.  FAIRMONTENSIS 
GROUP. 

*  Produce  pigment  on  gelatin  or  agar. 

t  Pigment  yellowish. 

Gelatin  liquefied.     Ps.  OCHRACEA  GROUP. 
Gelatin  not  liquefied.     Ps.  TURCOSA  GROUP. 
ft  Pigment  blue-violet. 

Gelatin  liquefied.     Ps.  JANTHINA  GROUP. 
Gelatin  not  liquefied.     Ps.  BEROLINENSIS  GROUP. 
**  Produce  a  greenish-bluish  fluorescence  in  culture-media. 

t  Gelatin  liquefied.    Ps.  PYOCYANEA  GROUP. 
ft  Gelatin  not  liquefied.     Ps.  SYNCYANEA  GROUP. 
2.  With  endospores,  aerobic  and  facultative  anaerobic. 

a.  Non-chromogenic. 

*  Rods  not  swollen  at  sporulation.     Ps.  ROSEA  GROUP. 
**  Rods    swollen    at    one    end    at    sporulation.     Ps.    TROMMEL- 

SCHLAGER  GROUP. 

b.  Produce  a  greenish-bluish  fluorescence  in  culture-media. 

*  Gelatin  liquefied.     Ps.  VIRIDESCENS  GROUP. 
**  Gelatin  not  liquefied.     Ps.  UNDULATA  GROUP. 

B.  Do  not  grow  in  nutrient  gelatin  or  other  organic  media.     NITRIMONAS 

GROUP. 

II.  Cell  plasma  with  a  reddish  tint,  also  with  sulphur  granules.     CHROMATIUM 
GROUP. 

MICROSPIRA  (Migula) 

I.  Cultures  show  a  bluish-silvery  phosphorescence.     PHOSPHORESCENT  GROUP. 
II.  Cultures  not  phosphorescent. 

A.  Gelatin  liquefied. 

1.  Cultures  show  the  nitro-indol  reaction. 

a.  Very  pathogenic  to  pigeons.     MSP.  METSCHNIKOVI  GROUP. 

b.  Not  distinctly  pathogenic  to  pigeons.     CHOLERA  GROUP. 

2.  Nitro-indol  reaction  negative  or  very  weak,  at  least  after  twenty- 

four  hours.     CHOLERA  NOSTRAS  GROUP. 

B.  Gelatin  not  liquefied  or  only  slightly  so.     MSP.  SAPROPHILA  GROUP. 

MYCOBACTERIUM  (Lehmann-Neumann) 

I.  Stain  with  basic  anilin  dyes,  and  easily  decolorized  by  mineral  acids  when 
stained  with  carbol-fuchsin. 


Chester's  Synopsis  of  Groups  of  Bacteria  233 

A.  Grow  well  on  nutrient  gelatin.     Gelatin  liquefied  very  slowly  or  merely 

softened. 

1.  Stain  by  Gram's  method.     SWINE  ERYSIPELAS  GROUP. 

2.  Not  stained  by  Gram's  method.     GLANDERS  GROUP. 

B.  Little  or  no  growth  in  ordinary  nutrient  gelatin. 

1.  Grow  well  in  nutrient  bouillon  at  body  temperatures. 

a.  Stained     by     Gram's     method.     Rods     cuneate — clavate — ir- 
regularly swollen.     DIPHTHERIA  GROUP. 

2.  No   growth   in   nutrient   bouillon   or  on    ordinary    culture-media. 

Rods  slender,  tubercle-like. 

a.  Stain  by  Gram's  method.     LEPROSY  GROUP. 

b.  Do  not  stain  by  Gram's  method.     INFLUENZA  GROUP. 

3.  No   growth   in   nutrient   bouillon   or   on    ordinary    culture-media. 

Rods  variable.     ROOT-TUBERCLE  GROUP. 

II.  Not  stained  with  aqueous  solutions  of  basic  anilin  dyes;  not  easily  decolorized 
by  acids.     TUBERCLE  GROUP. 

COCCACE.E 

Cells  in  their  free  condition  globular,  becoming  slightly  elongated  before  division. 
Cell  division  in  one,  two,  or  three  directions  of  space. 

A.  Cells  without  flagella. 

1.  Division  in  only  one  direction  of  space.     Streptococcus  (Billroth). 

2.  Division  in  two  directions  of  space.     Micrococcus  (Hallier). 

3.  Division  in  three  directions  of  space.     Sarcina  (Goodsir). 

B.  Cells  with  flagella. 

1.  Division  in  two  directions  of  space.     Planococcus  (Migula). 

2.  Division  in  three  directions  of  space.     Planosarcina  (Migula). 


CHAPTER  XII 
THE  BACTERIOLOGY  OF  THE  AIR 

MICRO-ORGANISMS  are  almost  universally  suspended  in  the  dust 
of  the  air,  their  presence  being  a  constant  source  of  contamination 
in  our  bacteriologic  researches  and  occasionally  a  menace  to  our 
health. 

Such  aerial  organisms  are  neither  ubiquitous  nor  uniformly 
disseminated,  but  are  much  more  numerous  where  the  air  is  polluted 
and  dusty  than  where  it  is  pure.  The  purity  of  the  atmosphere  bears 
a  distinct  relation  to  the  purity  of  the  surfaces  over  which  its  currents 
blow. 

The  micro-organisms  of  the  air  are  for  the  most  part  harmless 
saprophytes  taken  up  and  carried  about  by  the  wind.  They  are 
almost  always  taken  up  from  dry  materials,  experiment  having  shown 
that  they  arise  from  the  surf  aces  of  liquids  with  much  difficulty.  Not 
all  the  micro-organisms  of  the  air  are  bacteria,  and  a  plate  of  sterile 
gelatin  exposed  to  the  air  for  a  brief  time  will  generally  grow  molds 
and  6'idia  as  well. 

In  some  cases  the  bacteria  are  pathogenic,  especially  where  dis- 
charges from  diseased  animals  have  been  allowed  to  collect  and  dry. 
On  this  account  the  atmosphere  of  hospital  wards  and  of  rooms  in 
which  infectious  diseases  are  being  treated  is  more  apt  to  contain 
them  than  the  air  of  the  street.  However,  because  of  the  expectora- 
tion from  cases  of  tuberculosis,  influenza,  and  pneumonia,  which  is 
often  ejected  upon  the  sidewalks  and  floors  of  public  places,  the  pres- 
ence of  occasional  pathogenic  bacteria  is  far  from  uncommon  in 
street-dust. 

Giinther  points  out  that  the  greater  number  of  the  bacteria  which 
occur  in  the  air  are  cocci,  sarcina  being  particularly  abundant. 
Most  of  them  are  chromogenic  and  do  not  liquefy  gelatin.  It  is 
unusual  to  find  more  than  two  or  three  varieties  of  bacteria  at  a  time. 

To  determine  whether  bacteria  are  present  in  the  air  or  not,  all 
that  is  necessary  is  to  expose  a  film  of  sterile  gelatin  on  a  plate  or 
Petri  dish  to  the  air  for  a  while,  cover,  and  observe  whether  or  not 
bacteria  grow  upon  it. 

To  make  a  quantitative  estimation  is,  however,  more  difficult. 
Several  methods  have  been  suggested,  of  which  the  most  important 
may  be  briefly  mentioned: 

Hesse's  method  is  simple  and  good.  It  consists  in  making  a  measured 
quantity  of  the  air  to  be  examined  pass  through  a  horizontal  sterile  glass  tube 
about  70"  cm.  long  and  3.5  cm.  wide,  the  interior  of  which  is  coated  with  a  film 

234 


Sedgwick's  Method  235 

of  gelatin  in  the  same  manner  as  an  Esmarch  tube.  The  tube  is  closed  at 
both  ends  with  sterile  corks  carrying  small  glass  tubes  plugged  with  cotton. 
When  ready  for  use  the  tube  at  one  end  is  attached  to  a  hand-pump,  the  cotton 
removed  from  the  other  end,  and  the  air  slowly  passed  through,  the  bacteria  hav- 
ing time  to  sediment  upon  the  gelatin  as  they  pass.  When  the  required  amount 
has  passed,  the  tubes  are  again  plugged,  the  apparatus  stood  away  for  a  time, 
and  subsequently,  when  they  have  grown,  the  colonies  are  counted.  The 
number  of  colonies  in  the  tube  will  represent  pretty  accurately  the  number  of 
bacteria  in  the  volume  of  air  that  passed  through  the  tube. 

In  such  a  tube,  if  the  air  pass  through  with  proper  slowness,  the  colonies  will 
be  much  more  numerous  near  the  point  of  entrance  than  near  that  of  exit.  The 
first  to  fall  will  probably  be  those  of  heaviest  specific  gravity — i  e.,  the  molds. 

Petri's  Method. — A  more  exact  method  is  that  of  Petri,  who  uses  small  filters 
of  sand  held  in  place  in  a  wide  glass  tube  by  small  wire  nets.  The  sand 
used  is  made  to  pass  through  a  sieve  whose  openings  are  of  known  size,  is 
heated  to  incandescence,  then  arranged  in  the  tube  so  that  two  of  the  little  filters, 
held  in  place  by  their  wire-gauze  coverings,  are  superimposed.  One  or  both  ends 


Fig.  80. — Hesse's  apparatus  for  collecting  bacteria  from  the  air. 


of  the  tube  are  closed  with  corks  having  a  narrow  glass  tube.  The  apparatus 
is  sterilized  by  hot  air,  and  is  then  ready  for  use.  The  method  of  employment  is 
very  simple.  By  means  of  a  hand-pump  100  liters  of  air  are  made  to  pass 
through  the  filter  in  from  ten  to  twenty  minutes,  the  contained  micro-organisms 
being  caught  and  retained  by  the  sand.  The  sand  from  the  upper  filter  is  then 
carefully  mixed  with  sterile  melted  gelatin  and  poured  into  sterile  Petri  dishes, 
where  the  colonies  develop  and  can  be  counted.  Petri  points  out  in  relation  to 
his  method  that  the  filter  catches  a  relatively  greater  number  of  bacteria  in 
proportion  to  molds  than  the  Hesse  apparatus,  which  depends  upon  sedimenta- 
tion. Sternberg  points  out  that  the  chief  objection  to  the  method  is  the  presence 
of  the  sand,  which  interferes  with  the  recognition  and  counting  of  the  colonies 
in  the  gelatin. 

Sedgwick's  Method. — Sedgwick  and  Miquel  have  recommended  the  use  of 
a  soluble  material — granulated  or  pulverized  sugar — instead  of  the  sand.  The 
apparatus  used  for  the  sugar  experiments  differs  a  little  from  the  original  of  Petri, 
though  the  principle  is  the  same,  and  can  be  modified  to  suit  the  experimenter. 

A  particularly  useful  form  of  apparatus,  suggested  by  Sedgwick  and  Tucker, 
has  an  expansion  above  the  filter,  so  that  as  soon  as  the  sugar  is  dissolved  in  the 


236 


The  Bacteriology  of  the  Air 


melted  gelatin  it  can  be  rolled  out  into  a  film  like  that  of  an  Esmarch  tube. 
This  cylindric  expansion  is  divided  into  squares  which  make  the  counting  of  the 
colonies  very  easy. 

Roughly,  the  number  of  germs  in  the  atmosphere  may  be  estimated  at  from 
100  to  1000  per  cubic  meter. 

The  bacteriologic  examination  of  air  is  of  very  little  importance 
because  of  the  numerous  errors  that  must  be  met.  Thus,  when  the 
air  of  a  room  is  quiescent  it  may  contain  very  few  bacteria;  let  some 


Fig.  81.— Petri's  sand  filter  for  air- 
examination. 


Fig.  82. — Sedgwick  and  Tucker's  ex- 
panded tube  for  air-examination. 


one  walk  across  the  floor  so  that  dust  rises,  and  the  number  of  bac- 
teria becomes  considerably  increased;  if  the  room  be  swept,  the  in- 
crease is  enormous.  From  these  and  similar  contingencies  it  be- 
comes very  difficult  to  know  just  when  and  how  the  air  is  to  be  ex- 
amined, and  the  value  of  the  results  is  correspondingly  lessened. 

The  most  sensible  studies  of  the  air  aim  rather  at  the  discovery 
of  some  definite  organism  or  organisms  than  at  the  determination 
of  the  total  number  per  cubic  meter. 


CHAPTER  XIII 
BACTERIOLOGY  OF  WATER 

UNLESS  water  has  been  specially  sterilized,  and  received  and  kept 
in  sterile  vessels,  it  always  contains  some  bacteria,  the  number 
usually  bearing  a  distinct  relationship  to  the  quantity  of  organic 
matter  present. 

The  majority  of  the  water  bacteria  are  bacilli,  and  are  as  a  rule 
non-pathogenic.  Wright,*  in  his  examination  of  the  bacteria  of 
the  water  from  the  Schuylkill  River,  found  two  species  of  micrococci, 
two  species  of  cladothrices,  and  forty-six  species  and  two  varieties 
of  bacilli.  Pathogenic  bacteria,  such  as  the  spirillum  of  Asiatic 
cholera,  the  bacillus  of  typhoid  fever,  and  the  bacillus  of  dysentery 
may  occur  in  polluted  water,  but  are  exceptional. 

The  method  of  determining  the  number  of  bacteria  in  water  is  very 
simple,  and  can  be  accomplished  with  very  little  apparatus.  The 
method  depends  upon  the  equal  distribution  of  a  measured  quantity 


Fig.  83. — Wolfhugel's  apparatus  for  counting  colonies  of  bacteria  upon  plates. 

of  the  water  to  be  examined  in  some  sterile  liquefied  medium,  whose 
subsequent  solidification  in  a  thin  layer  permits  the  colonies  to  be 
counted. 

The  method  originated  with  Koch,  and  may  be  performed  with 
plates,  Petri  dishes,  or  Esmarch  rolls.  It  is  always  best  to  make  a 
number  of  cultures  with  different  quantities  of  the  water,  using,  for 
example,  o.oi,  o.i,  0.5,  and  i.o  cc.,  respectively,  to  a  tube  of  liquefied 
gelatin,  agar-agar,  or  glycerin  agar-agar. 

The  details  of  the  method  depend  upon  the  quality  of  the  water  to 
be  examined.  If  the  number  of  bacteria  per  cubic  centimeter  be 
small,  large  quantities  may  be  used;  but  if  there  be  millions  of  bac- 
teria in  every  cubic  centimeter,  it  may  be  necessary  to  dilute  the  water 
to  be  examined  in  the  proportion  of  i  :  10  or  i  :  100  with  sterile 
water,  mixing  well,  and  making  the  plate  cultures  from  the  dilutions. 

*  "Memoirs  of  the  National  Academy  of  Sciences,"  vol.  vn,  Third  Memoir. 

237 


238  Bacteriology  of  Water 

It  is  best  to  count  all  the  colonies  developed  upon  the  culture,  if 
possible;  but  when  hundreds  of  thousands  are  scattered  over  it,  an 
estimate  made  by  counting  and  averaging  the  number  in  each  of  the 
small  squares  of  some  counting  apparatus,  such  as  those  devised  by 
Wolfhiigel,  Esmarch,  or  Frost.  In  counting  the  colonies  a  lens  is 
indispensable. 

In  some  cases,  where  bacteria  are  exceedingly  numerous,  as  badly 
contaminated  waters,  in  the  study  of  sewage,  in  inflammatory  exu- 
dates,  and  in  cultures  intended  for  the  preparation  of  bacterial  vac- 
cines, it  is  expedient  to  directly  enumerate  the  bacteria  without 
resorting  to  the  cultivation  method, 
where  all  of  the  organisms  may  not  grow. 

Excellent  methods  for  the  computa- 
tion of  bacteria  have  been  devised.  That 
of  Winslow  and  Willcomb*  being  as 
follows : 


"The   cover-slips  should  be  boiled  in  a  10 
per  cent,  solution  of  potassium  bichromate  in 
50  per  cent,  sulphuric  acid  and  allowed  to  lie 
in   this  cleansing  mixture.     Just  before  using 
they   may   be   rinsed  in  50  per  cent,  alcohol 
and  dried  on  a  silk  cloth,  not  in  the  flame. 
One-twentieth  of  a  cubic  centimeter  of  water 
placed  on  such  a  cover-slip  spreads  evenly  and 
should  be  allowed  to  dry  in  the  air  without 
sudden  heating.     After  drying  it  is  fixed  by        Fig.  84. — Esmarch's   instru- 
passing  through  the  flame,  covered  with  Ziehl-     ment  for  counting  colonies  of 
Neelson's  carbol-fuchsin,  warmed  until  steam    bacteria  in  Esmarch  tubes, 
just  rises,  washed,  dried,  and  mounted.     For 

counting  the  bacteria  we  use  a  Sedgwick-Rafter  eye-piece  micrometer,  made 
for  the  study  of  the  larger  micro-organisms  in  drinking  water."  Very  uni- 
form results  have  followed. 

The  method  of  Wrightf  was  devised  for  the  computation  of  bac- 
teria in  suspensions  used  in  making  tests  of  the  opsonic  power  of  the 
blood  and  is  given  in  the  chapter  upon  "The  Opsonic  Index." 

The  majority  of  the  water  bacteria  rapidly  liquefy  gelatin,  on 
which  account  it  is  better  to  employ  both  gelatin  and  agar-agar  in 
making  the  cultures. 

In  ordinary  city  hydrant-water  the  bacteria  number  from  2  to 
50  per  cubic  centimeter;  in  good  pump- water,  100  to  500;  in  filtered 
water  from  rivers,  according  to  Giinther,  50  to  200;  in  unfiltered  river- 
water,  6000  to  20,000.  According  to  the  pollution  of  the  water  the 
number  may  reach  as  many  as  50,000,000. 

The  waters  of  wells  and  springs  are  dependent  for  their  purity 
upon  the  character  of  the  earth  or  rock  through  which  they  filter, 
and  the  waters  of  deep  wells  are  much  more  pure  than  those  of  shallow 
wells,  unless  contamination  take  place  from  the  surface  of  the 
ground. 

*  "Jour,  of  Infectious  Diseases,"  Supplement,  May,  1905,  No.  i,  p.  273. 
f  "Lancet,"  July  5,  1902. 


Determination  of  Bacteria  in  Water  239 

Ice  always  contains  bacteria  if  the  water  contained  them  before 
it  was  frozen.  In  Hudson  River  ice  Prudden  found  an  average  of 
398  colonies  in  a  cubic  centimeter. 

A  sample  of  water  when  collected  for  examination  should  be 
placed  in  a  clean  sterile  bottle  or  in  a  hermetically  sealed  pre-ster- 
ilized  glass  bulb,  and  must  be  examined  as  soon  as  possible,  as  the 
bacteria  multiply  rapidly  in  water  which  is  allowed  to  stand  for  a 
short  time.  If  the  water  to  be  examined  must  be  transported  any 
considerable  distance  before  the  manipulations  are  performed,  it 
should  be  packed  in  ice.  The  greatest  care  must  always  be  exercised 
that  the  unnatural  conditions  arising  from  the  bottling  of  the  water, 
the  changes  of  temperature,  and  the  altered  relationship  to  light  and 
the  atmosphere,  do  not  modify  the  number  of  contained  bacteria. 

1OO 


Fig.  85. — Frost's  plate  counter,  for  counting  colonies  of  bacteria  on  Petri 
dish  or  plate  cultures.  The  cross-lines  divide  the  figure  into  square  centimeters. 
The  numbers  at  the  top  of  the  figure  indicate  the  area  in  centimeters  of  the  various 
discs.  The  area  of  each  sector  (a  and  b)  is  one-tenth  of  the  whole  area. 

The  detection  of  such  important  bacteria  as  the  colon,  typhoid 
and  dysentery  bacilli,  and  the  cholera  spirillum,  will  be  considered 
in  the  chapters  treating  of  those  respective  organisms. 

Drinking-water,  especially  that  furnished  to  large  cities,  is  not 
infrequently  contaminated  with  sewage,  and  contains  intestinal 
bacteria — Bacillus  coli.  For  the  ready  determination  of  this  organ- 
ism, which  is  an  important  indication  that  the  water  is  polluted, 
Smith*  has  made  use  of  the  fermentation-tube  in  addition  to  the 
*  "Amer.  Jour.  Med.  Sci.,"  1895,  ex,  p.  301. 


240 


Bacteriology  of  Water 


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Determination  of  Bacteria  in  Water 


241 


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Zimmermann 
Schroter  .  .  . 

Zopf  
Frankland  .  . 

Flugge  .... 
Eisenberg  .  . 
Jager  .... 

Ehrenberg  .  . 
Frankland  .  . 

Jordan  .... 
Eisenberg  .  . 

Frankland  .  . 
Ravenel  .  .  . 

5  "Sb"Sb  to  B 

Escherich  .  . 
Escherich  .  . 

Eberth  .... 
Ravenel  .  .  . 
Weichselbaum 
Weichselbaum 

Frankland  .  . 

!  !  .  '. 

.   .   . 



Ul 

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B.  flavescens  .  .  . 
B.  lactis  ervthrogetu 
B.  subflavus  .  .  . 
Sarcina  lutea  .  .  . 

B.  janthinus  .  .  . 
B.  violaceus  .  .  . 

B.  mycoides  .  .  .  . 
B.  mesentericus  vul| 
"•  B.  proteus  fluoresce 

••  B.  subtilis  .... 
B.  cereus  

B.  cloacae  
B.  liquefaciens  .  . 

B.  liquidus  .... 
B.  antenniformis  . 

B.  superficialis  .  . 
B.  annulatus  .  .  . 
B.  flexuosus  .  .  . 
B.  geniculatus  .  . 
B.  aquatilis  commun 

—  B.  coli  communis 
B.  aerogenes  .  .  . 

B.  simultyphus  .  . 
B.  solitarius  .  .  . 
B.  aquatilis  sulcatus 
B.  aquatilis  sulcatus 

B.  candicans  .  .  . 

16 


242  Bacteriology  of  Water 

plate.  His  method  is  to  add  to  each  of  several  fermentation-tubes 
containing  i  per  cent,  dextrose-bouillon  a  certain  quantity  of  water. 
The  evolution  of  50-60  per  cent,  of  gas  by  the  third  day  is  a  strong 
indication  that  the  colon  bacillus  is  present.  The  presence  of  gas 
in  a  fermentation  tube  constitutes  the  "presumptive  test"  for  the 
colon  bacillus.  It  is  not  an  infallible  indication  of  their  presence. 
A  careful  study  of  its  usefulness  has  been  made  by  Ruediger  and 
Slyfield*  who  found  that  in  making  quantitative  determination  of 
B.  coli  in  polluted  waters  by  means  of  the  fermentation  tubes,  the 
most  accurate  results  were  obtained  by  the  use  of  neutral-red  lactose 
bouillon.  Gas  appeared  earlier  in  the  neutral-red  lactose  tubes 
than  in  lactose  bile  broth  tubes,  and  B.  coli  were  more  easily  isolated, 
by  plating,  from  the  former  than  from  the  latter.  The  finding  of 
B.  coli  in  the  fermentation  tubes  is  greatly  facilitated  by  making 
plates  soon  after  the  appearance  of  the  gas.  When  the  fermenta- 
tion of  the  sugar  and  the  appearance  of  gas  in  the  tube  occurs,  some 
bacteriologists  are  satisfied  that  B.  coli  are  present,  and  go  no  fur- 
ther, but  a  careful  workman  will  always  take  pains  to  confirm  the 
indications  of  their  presence  by  plating  and  isolating  the  bacillus  in 
pure  culture. 

It  was  at  one  time  thought  that  the  occurrence  of  the  colon  bacillus 
in  water  was  sufficient  to  condemn  its  potability,  but  the  evidence 
accumulated  in  recent  years,  showing  that  this  organism  may  reach 
streams  from  manured  soil,  may  enter  it  with  the  dejecta  of  domestic 
animals,  wild  animals,  birds,  and  perhaps  even  of  fishes,  makes  it 
doubtful  whether  anything  but  an  exceptionally  large  number  of  the 
organisms  should  be  looked  upon  as  indicative  of  sewage  pollution 
and  proof  that  the  water  is  not  potable. 

In  determining  the  species  of  bacteria  found  in  the  water  reference 
must  be  made  to  the  numerous  monographs  upon  the  subject  and  to 
special  tables.  An  excellent  table  of  this  kind,  arranged  by  Fuller,  f 
is  given  on  pages  240  and  241. 

Filtration  with  sand,  etc.,  diminishes  the  number  of  bacteria  for  a 
time,  but,  as  the  organisms  multiply  in  the  filter,  the  benefit  is  not 
permanent  and  the  filters  must  frequently  be  subjected  to  bacterio- 
logic  tests  and  the  sand  washed,  spread  out  to  dry  and  the  filters 
renewed.  Porcelain  filters  seem  to  be  the  only  positive  safeguard, 
and  even  these,  the  best  of  which  seems  to  be  the  Pasteur-Chamber- 
land,  allow  the  bacteria  to  pass  through  if  used  too  long  without  proper 
attention. 

For  those  whose  special  line  of  work  is  the  bacteriology  of  water, 
the  report  of  the  Committee  on  Standard  Methods  of  Water  Analysis 
to  the  Laboratory  Section  of  the  American  Public  Health  Association, 
published  in  Supplement  No.  i  of  the  "Journal  of  Infectious  Dis- 
eases," May,  1905,  will  prove  indispensable. 

*Jour.  of  the  American  Public  Health  Association,  1911,  i,  No.  11,  p.  828. 
t  "Public  Health  and  Journal  of  Experimental  Medicine." 


CHAPTER  XIV 
BACTERIOLOGY  OF  THE  SOIL 

THE  upper  layers  of  the  soil  contain  bacteria  in  proportion  to  their 
richness  in  organic  matter.  Near  the  habitations  of  men,  where  the 
soil  is  cultivated,  the  excrement  of  animals,  largely  made  up  of  bac- 
teria, is  spread  upon  it  to  increase  its  fertility,  this  treatment  not 
only  adding  new  bacteria  to  those  already  present,  but  also  enabling 
those  present  to  grow  much  more  luxuriantly  because  of  the  increased 
nourishment  they  receive. 

Where,  as  in  Japan,  human  excrement  is  used  to  fertilize  the  soil, 
or  as  in  India,  it  is  carelessly  deposited  upon  the  ground,  bacteria  of 
cholera,  dystentery ,  and  typhoid  fever  are  apt  to  become  disseminated 
by  fresh  vegetables,  or  through  water  into  which  the  soil  drains.  In 
such  localities  fresh  vegetables  should  not  be  eaten,  and  water  for 
drinking  should  be  boiled. 

The  researches  of  Flligge,  C.  Frankel,  and  others  show  that  the 
bacteria  of  the  soil  do  not  penetrate  deeply,  but  gradually  decrease 
in  number  until  the  depth  of  a  meter  is  reached,  then  rapidly  di- 
minish until  at  a  meter  and  a  quarter  they  rather  abruptly  disappear. 

The  bacteria  of  soil  are,  for  the  most  part,  harmless  saprophytes, 
though  a  few  highly  pathogenic  organisms,  such  as  the  bacilli  of 
tetanus  and  malignant  edema,  occur.  Many  of  them  are  anaerobic, 
and  it  is  interesting  to  speculate  upon  their  biology.  Whether  they 
develop  and  multiply  in  the  soil  in  intimate  association  with  strongly 
aerobic  organisms  by  which  the  free  oxygen  is  aborbed,  or  whether 
they  remain  latent  in  the  soil  and  develop  only  in  the  intestines  of 
animals,  is  not  known. 

The  estimation  of  the  number  of  bacteria  in  the  soil  seems  to  be 
devoid  of  any  practical  importance.  C.  Frankel  has,  however,  origi- 
nated an  accurate  method  of  determining  it.  By  means  of  a  special 
boring  apparatus  earth  can  be  secured  from  any  depth  without 
digging  and  without  danger  of  mixing  with  that  of  the  superficial 
strata.  A  measured  quantity  of  the  secured  soil  is  thoroughly 
mixed  with  liquefied  sterile  gelatin  and  poured  into  a  Petri  dish  or 
solidified  upon  the  walls  of  an  Esmarch  tube.  The  colonies  are 
counted  with  the  aid  of  a  lens.  Fliigge  found  in  virgin  earth  about 
100,000  colonies  in  a  cubic  centimeter. 

Samples  of  earth,  like  samples  of  water,  should  be  examined  as 
soon  as  possible  after  being  secured,  for,  as  Giinther  points  out,  the 
number  of  bacteria  changes  because  of  the  unusual  dryness,  warmth, 
exposure  to  oxygen,  etc. 

The  most  important  bacteria  of  the  soil  are  those  of  tetanus  and 

243 


244 


Bacteriology  of  the  Soil 


malignant  edema,  in  addition  to  which,  however,  there  are  a  great 
variety  of  organisms  pathogenic  for  rabbits,  guinea-pigs,  and  mice. 
In  the  "  Bacteriological  Examination  of  the  Soil  of  Philadelphia," 
Ravenel*  came  to  the  conclusion  that — 

1.  Made  soils,  as  commonly  found,  are  rich  in  organic  matter  and  excessively 
damp  through  poor  drainage. 

2.  They  furnish   conditions   more  suited   to   the   multiplication  of  bacteria 
than  do  virgin  soils,  unless  the  latter  are  contaminated  by  sewage  or  offal. 


Fig.  86. — Tip  of  Frankel's  instrument  tor  obtaining  earth  from  various 
depths  for  bacteriologic  study.  B  shows  the  instrument  with  its  cavity  closed, 
as  it  appears  during  boring;  A,  open,  as  it  appears  when  twisted  in  the  other 
direction  to  collect  the  earth. 

3.  Made  soils  contain  large  numbers  of  bacteria  per  gram  of  many  different 
species,  the  deeper  layers  being  as  rich  in  the  number  and  variety  of  organisms 
as  the  upper  ones.  After  some  years  the  number  in  the  deeper  layers  probably 
becomes  proportionally  less.  Made  soils  are  more  likely  than  others  to  contain 
pathogenic  bacteria. 

In  seventy-one  cultures  that  were  isolated  and  carefully  studied 
by  Ravenel,  there  were  two  cocci,  one  sarcina,  and  five  cladothrices; 
all  the  others  were  bacilli. 

*  "Memoirs  of  the  National  Academy  of  Sciences,"  First  Memoir,  1896. 


CHAPTER  XV 
THE  BACTERIOLOGY  OF  FOODS 

THE  relation  of  bacteria  to  foods  is  an  important  one  and  should 
be  as  thoroughly  understood  as  possible  by  both  the  profession  and 
the  laity.  The  relationship  may  be  expressed  thus: 

I.  Foods  serve  as  vehicles  by  which  infectious  agents  are  con- 
veyed to  the  body. 

II.  Foods  are  chemically  changed  and  made  unfit  for  use  by  the 
bacteria. 

I.  Foods  as  Fomites. — In  animal  food  the  first  source  of  infection 
is  the  animal  itself,  danger  of  infection  always  accompanying  the 
employment  of  foods  derived  from  diseased  animals.  Thus,  milk 
apparently  normal  in  appearance  has  been  found  to  contain  danger- 
ous pathogenic  bacteria.  The  tubercle  bacillus  is  one  of  the  most 
important  of  these,  and  at  the  present  time  the  consensus  of  opinion 
inclines  toward  the  view  that  the  great  prevalence  of  tuberculosis 
among  human  beings  depends  partly  upon  the  ingestion  of  tubercle 
bacilli  in  milk.  It  does  not  appear  necessary  that  the  udder  of  the 
cow  be  diseased  in  order  that  the  organisms  enter  the  milk,  as  they 
seem  to  have  been  found  in  milks  derived  from  cows  whose  udders 
were  entirely  free  from  demonstrable  tuberculosis.  It  is,  therefore, 
imperative  to  retain  only  healthy  cows  in  the  dairy,  and  careful 
legislation  should  provide  for  the  detection  and  destruction  of  all 
tuberculous  animals.  The  detection  of  tubercle  bacilli  in  milk  can 
only  be  certainly  accomplished  by  the  injection  of  a  few  cubic  centi- 
meters of  the  fluid  into  guinea-pigs  and  noting  the  results. 

In  addition  to  the  tubercle  bacillus,  pyogenic  streptococci  have 
been  observed  in  enormous  quantities  and  almost  pure  culture  in 
milk  drawn  from  cows  suffering  from  mastitis.  Stokes*  has  observed 
a  remarkable  case  of  this  kind  in  which  the  milk  contained  so  much 
pus  that  it  floated  upon  the  top  like  cream.  Such  seriously  in- 
fected milk  could  not  be  used  with  safety  to  the  consumer. 

In  market  milk  one  occasionally  finds  pathogenic  organisms,  such 
as  the  diphtheria  bacillus,  typhoid  bacillus,  streptococcus,  etc.,  de- 
rived from  human  sources.     Such  polluted  milks  have  been  known 
to  spread  epidemics  of  the  respective  diseases  whose  micro-organisms 
are  present.     Bacteria  may  enter  milk  from  careless  handling,  from 
water  used  to  wash  the  cans  or  to  dilute  the  milk,  or  from  dust;  and 
as  milk  is  an  excellent  medium  for  the  growth  of  bacteria,  it  should 
*  "Maryland  Medical  Journal,"  Jan.  9,  1897. 
245 


246  The  Bacteriology  of  Foods 

always  be  treated  with  the  greatest  care  to  prevent  such  contamina- 
tion, as  saprophytic  bacteria  produce  chemical  changes  in  the  milk, 
such  as  acidity  and  coagulation,  which  destroy  its  usefulness  or 
render  it  dangerous  as  food  for  infants  and  invalids.  Where  the 
necessary  precautions  are  not  or  cannot  be  taken,  Pasteurization  of 
the  milk  as  soon  after  its  reception  as  possible  may  act  as  a  safeguard. 

The  student  interested  in  the  sanitary  relations  of  milk  cannot 
do  better  than  refer  to  Bulletin  No.  35  of  the  Hygienic  Laboratory, 
Washington,  D.  C.,  1907,  "Upon  the  Origin  and  Prevalence  of 
Typhoid  Fever  in  the  District  of  Columbia,"  and  to  Bulletin  No.  41 
of  the  same  laboratory,  upon  "Milk  and  its  Relation  to  the  Public 
Health"  (1908);  also  to  the  "Bacteriology  of  Milk,"  by  Swithinbank 
and  Neuman,  New  York,  E.  P.  Dutton  &  Co.,  1903. 

Meat  from  tuberculous  animals  might  cause  disease  if  eaten  raw 
or  but  partially  cooked.  As  cooking  suffices  to  kill  the  organisms, 
the  danger  under  ordinary  conditions  is  not  great.  Moreover, 
tuberculosis  rarely  affects  the  muscles,  the  parts  usually  eaten. 

Butter  made  from  cream  derived  from  tuberculous  milk  may  also 
contain  tubercle  bacilli,  as  has  been  shown  by  the  researches  of 
Rabinowitsch.* 

Foods  may  become  polluted  with  bacteria  in  a  variety  of  ways 
that  will  suggest  themselves  to  the  reader.  The  common  source  is 
dust,  which  is  more  or  less  rich  in  bacteria  according  to  the  soil  from 
which  it  arises.  The  readiness  with  which  raw  foods,  such  as  meats, 
milk,  etc.,  can  be  thus  contaminated  in  the  barnyard,  dairy,  slaughter- 
house, and  shop,  teaches  but  one  lesson — that  the  greatest  cleanli- 
ness should  prevail  for  the  sake  of  the  dealer,  whose  goods  may  be 
spoiled  by  carelessness,  and  the  consumer,  who  may  be  injured  by 
the  food. 

Any  food  may  carry  infectious  organisms  upon  its  surface,  such 
organisms  being  derived  from  the  hands  of  the  dealer,  from  dust, 
from  water,  as  when  green  vegetables  are  sprinkled  with  impure 
water  to  keep  them  fresh,  or  from  other  sources. 

The  cleanliness  of  the  merchant  and  the  protection  from  contami- 
nation that  he  bestows  upon  his  goods  should  be  taken  into  consid- 
eration by  his  customers. 

Shell-fish,  especially  oysters,  seem  to  be  common  carriers  of  infec- 
tion, especially  of  typhoid  fever.  The  oysters  seem  to  be  contami- 
nated with  infected  sewage  carried  to  their  beds.  It  is  not  yet 
satisfactorily  determined  whether  typhoid  bacilli  multiply  in  the 
juices  in  the  shells  of  the  oysters  or  not,  but  a  number  of  epidemics 
of  typhoid  fever  have  been  very  conclusively  traced  to  the  consump- 
tion of  certain  oysters  at  a  definite  time  and  place.  As  cooking  the 
oysters  will  kill  the  contained  bacilli,  the  prophylaxis  of  disease  in 
this  case  is  very  simple. 

*"  Deutsche  med.  Wochenschrift,"  1900,  No.  26;  abstract  in  the  "Centralbl. 
f.  Bakt.,"  etc.,  1901,  xxix,  p.  309. 


Food  Poisons  247 

II.  Food  Poisons. — The  nomenclature,  suggested  by  Vaughan  and 
Novy,*  contains  the  following  terms: 

Bromatotoxism — food-poisoning ; 
Galactotoxism — milk-poisoning ; 
Tyrotoxism — cheese-poisoning ; 


JL.  yi  ui/VA/i>-3 /rit        v-iiu^3c;-|j*_»io\Jiiiii^  , 

Kreotoxism — meat-poisoning ; 
Ichthyotoxism — fish-poisoning ; 
Mytilotoxism — mussel-poisoning; 
Sitotoxism — cereal-poisoning. 


The  most  important  chemic  alterations  effected  by  bacteria  occur 
in  milk  and  meat. 

1.  Milk-poisoning    (Galactotoxism). — Milk,    even    when    freshly 
drawn  from  the  cow,  always  contains  some  bacteria,  whose  numbers 
gradually  diminish  for  a  few  hours,   then  rapidly  increase   until 
almost   beyond   belief.     These   organisms  are  for   the   most   part 
harmless  to  the  consumer,  but  ultimately  ruin  the  milk.     Although 
much  attention  has  been  paid  to  the  subject,  bacteriologists  are  not 
agreed  whether  the  number  of  bacteria  contained  in  milk  is  a  satis- 
factory guide  as  to  its  harmfulness. 

The  poisonous  change  in  milk,  cream,  ice-cream,  etc.,  has  been 
shown  by  Vaughan  to  depend  in  part  upon  the  presence  of  a  ptomain 
known  as  tyrotoxicon,  formed  by  the  growth  of  bacteria  in  the  milk, 
but  whether  by  any  particular  bacterium  is  not  known.  The  milk 
may  become  poisonous  during  any  time  of  the  year,  but  chiefly  in 
the  summer,  when,  because  of  the  higher  temperature,  bacteria 
develop  most  rapidly.  The  change  takes  place  in  stale  milk,  and 
it  is  supposed  that  many  cases  of  what  was  formerly  looked  upon  as 
" summer  complaint"  in  infants  were  really  poisoning  by  this  toxic 
ptomain. 

Ice-cream  poisoning  depends  upon  the  growth  of  the  bacteria  in 
the  milk  before  it  is  frozen.  In  some  cases  the  error  made  has  been 
to  prepare  the  cream  for  freezing  and  then  keep  or  transport  it,  the 
freezing  operation  being  delayed  until  the  development  of  the  bac- 
teria has  led  to  the  poisonous  condition. 

Cheese- poisoning  (Tyrotoxism)  is  also  thought  to  depend  upon 
tyrotoxicon  at  times,  though  it  has  been  shown  that  other  cheese 
poisons  exist.  It  is  more  or  less  a  question  whether  cases  of  milk- 
and  cheese-poisoning  do  not  depend  upon  the  toxic  products  of  the 
colon  bacillus  growing  in  the  foods. 

2.  Meat-poisoning   (Kreotoxism). — Botulism   or    meat-poisoning 
depends  upon  the  growth  of  certain  bacteria,  Bacillus  botulinus  of 
van  Ermengem,f  in  the  meat.     The  symptoms  following  infection 
by  the  organism  sometimes  closely  resemble  those  of  typhoid  fever, 
and  are  characterized  by  acute  gastro-intestinal  irritation,  nervous 

*  "Cellular  Toxins,"  Phila.,  1902. 

f  "Zeitschrift  fur  Hygiene,"  Bd.  xxvi,  Heft  i. 


248  The  Bacteriology  of  Foods 

disturbances,  and,  in  case  of  death,  by  fatty  degenerations  in  the 
organs  and  minute  interstitial  hemorrhages. 

3.  Fish-poisoning    (Ichthyotoxism)    sometimes   follows   the   con- 
sumption of  canned  and  presumably  spoiled  fish,  sometimes  the 
consumption  of  diseased  fish.     It  is  not  known  whether  it  depends 
upon  ptomains  or  upon  toxicogenic  germs,  though  probably  the 
latter,  as  Silber  has  isolated  a  Bacillus  piscicidus  that  is  highly 
toxicogenic. 

4.  Mussel-poisoning   (Mytilotoxism)   depends  partly   upon   irri- 
tating and  nervous  poisons  in  the  mussel  substance,  in  part  upon 
toxicogenic  germs  that  they  harbor. 

5.  Canned  Goods. — Improperly  preserved  canned  goods  not  in- 
frequently spoil  because  of  the  growth  of  bacteria,  but  the  occur- 
rence of  gas-formation,  acidity,  insipidity,  etc.,  causes  rejection  of 
the  product,  and  but  few  cases  of  supposed  poisoning  from  canned 
goods  can  be  authenticated. 


CHAPTER  XVI 

THE  DETERMINATION  OF  THE  THERMAL  DEATH-POINT 

OF  BACTERIA 

SEVERAL  methods  may  be  employed  for  this  purpose.  Roughly, 
it  may  be  done  by  keeping  a  bouillon  culture  of  the  micro-organism 
to  be  investigated  in  a  water-bath  whose  temperature  is  gradually 
increased,  transplantations  being  made  from  time  to  time  until  the 
fatal  temperature  is  reached. 

It  is  economy  to  make  the  transplantations  less  frequently  at 
first  than  later  in  the  experiment,  when  the  ascending  temperature 
approaches  a  height  dangerous  to  life.  In  ordinary  determinations 
it  is  well  to  make  a  transfer  at  4o°C.,  another  at  45°,  another  at  50°, 
still  another  at  55°,  and  then,  beginning  at  60°,  make  one  for  every 
additional  degree.  The  day  following  the  experiment  it  will  be  ob- 
served that  all  the  cultures  grow  except  those  heated  beyond  a 
certain  point,  say  62°C.,  when  it  can  properly  be  concluded  that 
62°C.  is  the  thermal  death-point.  If  all  the  transplantations  grow, 
of  course  the  maximum  temperature  was  not  reached,  and  the  ex- 
periment must  be  repeated  and  the  bacteria  exposed  to  still  higher 
temperatures. 

When  more  accurate  information  is  desired,  and  one  wishes  to 
know  how  long  the  micro-organism  can  endure  some  such  tempera- 
ture as  6o°C.  without  losing  its  vitality,  a  dozen  or  more  bouillon- 
tubes  may  be  inoculated  with  the  organism  to  be  studied,  and  stood 
in  a  water-bath  kept  at  the  temperature  to  be  investigated.  The 
first  can  be  removed  as  soon  as  it  is  heated  through,  another  in  five 
minutes,  another  in  ten  minutes,  or  at  whatever  intervals  the  thought 
and  experience  of  the  experimenter  shall  suggest,  the  subsequent 
growth  in  each  culture  showing  that  the  endurance  of  the  organism 
had  not  yet  been  exhausted.  By  using  gelatin,  pouring  each 
culture  into  a  Petri  dish,  and  subsequently  counting  the  colonies,  it 
can  be  determined  whether  many  or  only  a  few  of  the  organisms  in  a 
culture  possess  the  maximum  resisting  power.  To  determine  the 
percentage,  it  is  necessary  to  know  how  many  bacteria  were  present 
in  the  tubes  before  exposure  to  the  destructive  temperature.  'Ap- 
proximately the  same  number  can  be  placed  in  each  tube  by  adding 
the  same  measured  quantity  of  a  fluid  culture  to  each. 

In  both  of  the  procedures  one  must  be  careful  that  the  temperature 
of  the  fluid  in  the  test-tube  is  identical  with  that  of  the  water  in  the 
bath.  A  sterile  thermometer  introduced  into  an  uninoculated  tube 

249 


250  The  Thermal  Death-point 

exposed  under  conditions  similar  to  those  of  the  experiment  can  be 
used  as  an  index  for  the  others. 

Another  method  of  accomplishing  the  same  end  is  by  the  use  of 
Sternberg's  bulbs.  These  are  small  glass  bulbs  blown  on  one  end 
of  a  glass  tube,  drawn  out  to  a  fine  point  at  the  opposite  end.  If 
such  a  bulb  be  heated  so  that  the  air  is  expanded  and  partly  driven 
out,  its  open  tube,  dipped  into  inoculated  bouillon,  will  in  cooling 
draw  the  fluid  in,  so  as  to  fill  it  one-third  or  one-half.  A  number  of 
these  tubes  are  filled  in  this  manner  with  a  freshly  inoculated  culture 
medium  and  then  floated,  tube  upward,  upon  a  water-bath  whose 
temperature  is  gradually  elevated,  the  bulbs  being  removed  from 
time  to  time  as  the  required  temperatures  are  reached.  As  the 
bulbs  are  already  inoculated,  all  that  is  necessary  is  to  stand  them 
aside  for  a  day  "or  two,  and  observe  whether  or  not  the  bacteria 
grow,  determining  the  death-point  exactly  as  in  the  other  case. 


CHAPTER  XVII 

DETERMINATION  OF  THE  VALUE  OF  ANTISEPTICS, 
GERMICIDES,  AND  DISINFECTANTS 

THE  student  must  bear  in  mind  that  an  antiseptic  is  a  substance 
capable  of  restraining  the  growth  of  bacteria;  a  germicide,  one  ca- 
pable of  killing  them.  All  germicides  are  antiseptic  in  dilute  solu- 
tions, but  not  all  antiseptics  are  germicides.  Disinfectants  must  be 
germicides. 

Antiseptics  are  chiefly  employed  for  purposes  of  preservation, 
and  are  largely  used  in  the  industries  to  protect  organic  substances 
from  the  micro-organisms  of  fermentation  and  decomposition.  The 
problem  is  to  secure  a  satisfactory  effect  with  the  addition  of  the 
least  possible  preservative  in  order  that  its  presence  shall  not  chem- 
ically destroy  the  good  qualities  of  the  substances  preserved.  In 
the  case  of  foods  it  becomes  necessary  to  use  preservatives  free  from 
poisonous  properties. 

Disinfectants  and  germicides  are  employed  for  the  purpose  of 
destroying  germs  of  all  kinds,  and  the  chief  problem  is  to  secure 
efficiency  of  action,  rather  than  to  endeavor  to  save  on  the  reagent, 
which  would  be  a  false  economy,  in  that  the  very  object  desired  might 
be  defeated. 

The  following  methods  of  determining  the  antiseptic  and  germi- 
cidal  values  of  various  agents  can  be  elaborated  according  to  the 
extent  and  thoroughness  of  the  investigation  to  be  made. 

I.  The  Antiseptic  Value. — Remembering  that  an  antiseptic  is  a 
substance  that  inhibits  bacterial  growth,  the  determination  of  its 
value  can  be  made  by  adding  varying  quantities  of  the  antiseptic 
to  be  investigated  to  culture-media  in  which  bacteria  are  subse- 
quently planted.  It  is  always  well  to  use  a  considerable  number  of 
tubes  of  bouillon  containing  varying  strengths  of  the  reagent  to  be 
investigated.  If  the  antiseptic  be  non-volatile,  it  may  be  added 
before  sterilization,  which  is  to  be  preferred;  but  if  volatile,  it  must 
be  added  by  means  of  a  sterile  pipet,  with  the  greatest  precaution 
as  regards  asepsis,  after  sterilization  and  immediately  before  the  test 
is  made.  Control  experiments — i.e.,  bouillon  cultures  without  the 
addition  of  the  antiseptic — should  always  be  made. 

The  results  of  antiseptic  action  are  two:  retardation  of  growth  and 
complete  inhibition  of  growth.  As  the  inoculated  tubes  containing 
the  antiseptic  are  watched  in  their  development,  it  will  usually  be 
observed  that  those  containing  very  small  quantities  develop  al- 
most as  rapidly  as  the  control  tubes;  those  containing  more,  a  little 

251 


252 


Value  of  Antiseptics 


more  slowly;  those  containing  still  more,  very  slowly,  until  at  last 
there  comes  a  time  when  the  growth  is  entirely  checked. 

Sternberg  points  out  that  the  following  conditions,  which  must  be 
avoided,  may  modify  the  results  of  experiment: 

1.  The  composition  of  the  nutrient  media,  with  which  the  anti- 
septic may  be  incompatible  (as  bichloride  of  mercury  and  albumin). 

2.  The  nature  of  the  test-organism,  no  two  organisms  being  ex- 
actly alike  in  their  susceptibility. 

3.  The  temperature  at  which  the  experiment  is  conducted,  a 


C 


Same  rod  immersed  in  broth  after 
exposure  to  disinfectant. 

Fig.  87. — Glass  rod  in  test-tube,  for  use  in  testing  disinfectants.  Tube 
6  in.  by  %  in.;  rod  9  in.  by  y±  in.  Ring  marked  with  diamond  i  in.  from  lower 
end,  to  show  upper  limit  of  area  on  which  the  organisms  are  dried.  After  ex- 
posure the  rod  is  placed  in  a  similar  tube  containing  broth,  to  test  development, 
a,  Cotton  plug  wrapped  around  glass  rod;  b,  broth;  c,  gummed  label  on  handle 
of  rod,  for  identification;  d,  ring  marked  by  diamond;  e,  dried  organisms. 

relatively  greater  amount  of  the  antiseptic  being  necessary  at  tem- 
peratures favorable  to  the  organism  than  at  temperatures  unfavorable. 

4.  The  presence  of  spores  which  are  always  more  resistant  than  the 
asporogenous  forms. 

II.  The  Germicidal  Value. — Koch's  original  method  of  determin- 
ing this  was  to  dry  the  micro-organisms  upon  sterile  threads  of  linen 
or  silk,  and  then  soak  them  for  varying  lengths  of  time  in  the  germi- 
cidal  solution.  After  the  bath  in  the  reagent  the  threads  were  washed 
in  clean,  sterile  water,  transferred  to  fresh  culture-media,  and  their 


Testing  Germicidal  Value  of  Liquids  253 

growth  or  failure  to  grow  observed.  This  method  also  determines 
the  time  in  which  a  certain  solution  will  kill  micro-organisms,  so  is 
advantageous. 

Sternberg  suggested  a  method  by  which  the  dilution  necessary  to 
kill  the  bacteria  could  be  determined,  the  time  remaining  constant 
(two  hours'  exposure)  in  all  cases.  "Instead  of  subjecting  test- 
organisms  to  the  action  of  the  disinfecting  agent  attached  to  a  silk 
thread,  a  certain  quantity  of  a  recent  culture — usually  5  cc. — is 
mixed  with  an  equal  quantity  of  a  standard  solution  of  the  germi- 
cidal  agent,  .  .  .  and  after  two  hours'  contact  one  or  two  loopfuls 
are  transferred  to  a  suitable  nutrient  medium  to  test  the  question 
of  disinfection." 

A  very  simple  and  popular  method  of  determining  the  germicidal 
value  is  to  make  a  series  of  dilutions  of  the  reagent  to  be  tested ;  add 
to  each  a  small  quantity  of  a  fresh  liquid  culture,  and  at  varying  in- 
tervals of  time  transfer  a  loopful  to  fresh  culture-media.  By  a  little 
ingenuity  this  method  may  be  made  to  yield  information  as  to  both 
time  and  strength. 

Hill*  has  suggested  a  convenient  method  of  handling  the  cul- 
tures, which  are  dried  upon  the  ends  of  sterile  glass  rods  and  can  then 
be  transferred  from  one  solution  to  another  or  otherwise  manipulated. 

The  Modern  Method  of  Testing  the  Germicidal  Value  of  Liquids.-^- 
The  methods  of  testing  germicidal  strength  given  above  are  uncertain 
and  inaccurate,  and  can  only  be  looked  upon  as  "rough  and  ready" 
methods,  that  should  be  willingly  abandoned  for  anything  better. 
Three  methods  are  now  offered  that  hold  out  the  promise  of  scientific 
accuracy  through  an  established  standard  of  comparison.  In  the  order 
of  their  appearance,  which  is  also,  probably,  the  order  of  their  impor- 
tance, these  are  the  method  of  Rideal  and  Walker,f  "The  Lancet 
Method, "{  and  the  method  of  Anderson  and  McClintic.§  The 
methods  are  similar  in  general  principles,  and  have  the  same  object 
in  view,  i.e.,  the  expression  of  the  germicidal  value  of  any  sub- 
stance as  the  carbolic  acid  or  phenol  "coefficient."  Experience 
with  the  methods  leads  to  the  conviction  that  the  Rideal  and  Walker 
method  is  the  more  easy  to  execute,  but  that  the  Anderson-McClintic 
method  is  the  more  accurate.  As  the  latter  in  addition  to  its  ac- 
curacy has  now  become  the  standard  method  of  the  United  States 
Government,  it  is  the  method  with  which  the  student  should  be 
acquainted  and  which  will  be  given  in  detail. 

I.  The  Apparatus,  Reagents,  etc.,  Required  for  the  Test. — i.  A 
Phenol  Solution  that  shall  act  as  the  standard  of  comparison.  In  the 
preparation  of  this  solution,  pure  phenol — as  free  from  cresols,  etc., 
as  possible — should  be  employed.  Walker  recommends  that  only 

*  "Public  Health,"  vol.  xxiv,  p.  246. 

t  Journal  of  the  Royal  Sanitary  Institute,  London,  1903,  p.  424. 
t  "The  Standardization  of  Disinfectants"  (unsigned  article) ,  Lancet,  London, 
vol.  CLXXVII,  Nos.  4498,  4499  and  4500. 

§  Bulletin  No.  82  of  the  Hygienic  Laboratory,  Washington,  D.  C.,  1912. 


254  Value  of  Antiseptics 

phenol  with  a  melting  point  of  4o.5°C.,  be  used,  as  only  such  is  entirely 
free  from  impurities.  The  Eighth  Revision  of  the  U.  S.  Pharma- 
copoeia declares  phenol  with  a  melting  point  of  4o°C.  to  be  pure  and 
that  is  the  quality  that  may  be  accepted  as  the  standard. 

The  phenol  used  at  the  Hygienic  Laboratory  is  Merck's  "Silver 
Label."  The  standard  dilution,  made  by  the  U.  S.  P.  method 
(Koppeschaar),  contains  exactly  5  per  cent,  of  pure  phenol  by  weight, 
in  distilled  water.  From  this  stock  solution,  the  higher'dilutions  are 
made  fresh  each  day  for  that  day's  tests. 

2.  The  Solution  to  be  Tested. — A  5  per  cent,  solution  is  made  by 
adding  5  cc.  of  the  disinfectant  to  95  cc.  of  sterile  distilled  water 
with  a  standardized  5  cc.  capacity  pipet.     After  filling  the  pipet, 
all  excess  of  the  disinfectant  on  its  outside  is  wiped  off  with  sterile 
gauze.     The  contents  of  the  pipet  are  then  delivered  into  a  cyl- 
inder containing   95    cc.  of   sterile  distilled   water  and   the   pipet 
washed  out  as  clean  as  possible  by  aspiration  and  blowing  out  the 
contents  into  the  cylinder.     The  contents  of  the  cylinder  are  then 
thoroughly  shaken. 

3.  The  Test  Organism  selected  is  Bacillus  typhosus.     Before  be- 
ginning the  tests,  the  organisms  in  bouillon  culture  should  be  trans- 
planted to  fresh  media  every  twenty-four  hours  for  at  least  three 
successive  days.     In  making  the  transfers  one  loopful  of  a  4-mm. 
platinum  loop  is  carried  over.     In  exposing  the  culture  to  the  dis- 
infectant, Jf  0  cc.  of  the  culture  is  always  added  to  5  cc.  of  the 
diluted  disinfectant,  the  amount  being  measured  by  pipets  graduated 
in  tenths  of  a  cubic  centimeter. 

4.  The  Inoculating  Loops. — These  loops  are  made  of  No.  23  U.  S. 
standard  gauge  platinum  wire,  each  loop  being  4  mm.  in  diameter. 
There  should  be  four,  and  preferably  six,  such  loops  mounted  in 
the  usual  glass  handles,  ready  for  use.     In  order  to  facilitate  their 
sterilization,  a  special  holder  is  used. 

5.  The   Water-bath. — As   variations  in   the   temperature   of   the 
disinfecting  solutions  hasten  or  retard  their  destructive  action,  a 
temperature  of  2o°C.  has  been  arbitrarily  adopted  as  the  standard. 
For  its  maintenance  the  following  simply  constructed  water-bath 
has  been  devised.     It  consists  of  a  wooden  box  20  inches  deep,  21 
inches  long  and  21  inches  wide.     Inside  this  box  a  i4-quart  agate- 
ware pail,  10  inches  deep,  is  placed  and  saw-dust  is  well  packed 
around,  sufficient  being  placed  in  the  bottom  of  the  box  to  bring 
the  rim  of  the  pail  on  a  level  with  the  top  of  the  box.     A  tightly 
fitting  wooden  cover,  so  made  that  the  edges  project  slightly  over 
the  rim,  is  placed  over  the  pail.     In  the  cover  are  a  sufficient  number 
of  holes  for  the  seeding  tubes,  a  thermometer,  and  the  tube  contain- 
ing the  culture.     About  3   inches  below   the  rim  of  the  pail  a 
false  bottom  of  wire  gauze  is  placed;  this  is  for  the  seeding  tubes, 
etc.,  to- rest  on;     Water  is  placed  in  the  pail  to  within  half  an  inch 
of  the  top.     When  an  experiment  is  about  to  be  made  the  tempera- 


Testing  Germicidal  Value  of  Liquids 


255 


ture  of  the  water  in  the  pail  is  taken,  and  if  above  or  below  2O°C., 
it  is  brought  to  the  desired  temperature  by  the  addition  of  either 


Fig.  88. — Water-bath  showing  position  of  holes  for  seeding  tubes  and  ther- 
mometer in  place  (Anderson  and  McClintic,  in  Bulletin  No.  82,  Hygienic 
Laboratory). 


Fig.  89. — Cross  section  of  water-bath  showing  seeding  tubes  in  place  (Ander- 
son and  McClintic,  in  Bulletin  No.  82,  Hygienic  Laboratory). 

hot  or  cold  water.  When  the  proper  temperature  has  thus  been 
adjusted,  very  little  change  takes  place  in  an  hour's  time.  The 
apparatus  is  shown  in  the  cut. 


256  Value  of  Antiseptics 

6.  The  Culture-media  used  for  the  primary  culture,  and  for  the 
subcultures,  made  after  exposure  of  the  micro-organism  to  the  dis- 
infectant, is  nutrient  bouillon  made  with  Leibig's  beef  extract  in 
the  usual  manner  and  given  a  reaction  of  exactly  +  1.5.     Anderson 
and  McClintic  achieve  this  by  so  carrying  out  the  titrating  of  the 
medium  that  a  distinctly  perceptible  pink  color  marks  the  point  at 
which  the  addition  of  the  alkali  stops  (see  directions  for  titrating 
culture-media). 

7.  The  Tubes  for  the  culture  and  subcultures  are  ordinary  culture 
tubes,  containing  5  cc.  of  the  nutrient  bouillon  mentioned  above. 
They  are  filled,  plugged  and  sterilized  in  the  usual  manner. 

The  tubes  for  "seeding,"  i.e.,  exposing  the  bacteria  to  the  ger- 
micide, are  more  convenient  when  shorter.  At  the  time  of  transfer, 
the  platinum  loop  is  to  be  introduced  into  the  tube  as  it  stands  in 
the  water-bath  and  as  this  is  not  easy  with  tubes  of  standard  length, 
Anderson  and  McClintic  recommend  tubes  i  inch  in  diameter  and 
3  inches  long.  These  are  plugged  and  sterilized  by  dry  heat, 
or  as  recommended  by  the  authors  quoted,  are  sterilized  mouth 
down,  without  plugs  in  a  paper-lined  wire  basket. 

8.  The  Dilution  of  the  Phenol  and  Test  Solutions. — This  is  done  in 
standardized    graduates   with   standardized   pipets,    according    to 
the  requirements  of  the  particular  case.     Anderson  and  McClintic 
give  tables  that  are  useful  for  making  the  dilutions,  though  with  the 
aid  of  a  little  arithmetic  it  is  easy  to  calculate  the  proportions  of 
the  5  per  cent,  solutions  already  prepared,  and  sterile  distilled  water 
necessary  to  make  the  test  solutions  required.     As  it  is  certain  that 
some  of  the  dilutions  will  be  below  germicidal  strength,  and  as 
" weeds"  may  be  more  difficult  to  kill  than  the  test  organism  (B. 
typhosus)  it  is  important  to  see  that  the  distilled  water  used  for 
dilution  is  sterile,  and  that  the  cylinders  and   bottles  or  pipets 
used  for  making  the  dilutions  are  all  sterile  and  that  the  dilutions 
themselves  are  made  with  aseptic  precautions. 

Under  the  standard  conditions  recommended,  the  phenol  solu- 
tion that  destroys  all  of  the  B.  typhosus  introduced,  in  2%  minutes 
is  i  :  80,  but  it  is  always  wise  to  make  additional  dilutions  to  control 
the  strength,  as  shown  in  the  table  below.  When  the  strength 
of  the  disinfectant  or  germicide  to  be  tested  is  entirely  unknown, 
it  is  well  to  begin  by  making  a  number  of  tests  with  widely  sepa- 
rated dilutions,  by  one  of  the  "rough  and  ready"  methods,  so  as 
to  arrive  at  an  approximate  strength,  before  commencing  the  more 
difficult  technic  required  for  the  determination  of  the  phenol  co- 
efficient, which  should  be  looked  upon  as  the  final  test  for  exact 
comparison. 

9.  Racks  for  Holding  the  Tubes  are  indispensable.     The  "seeding 
tubes,"  that  is,  the  tubes  in  which  the  actual  exposure  of  the  culture 
to  the  germicidal  solutions  is  to  take  place,  have  already  been  pro- 
vided for  in  the  construction  of  the  water-bath. 


Testing  Germicidal  Value  of  Liquids 


257 


For  the  " subculture"  tubes,  any  test-tube  rack  will  do,  but  it 
is  more  convenient  to  have  a  special  rack  or  stand  made.  That 
recommended  contains  five  rows  of  14  holes  each.  Each  tube  of 
culture-medium  is  carefully  marked  with  a  blue  pencil  to  show  three 
things,  i,  the  germicide;  2,  the  dilution;  3,  the  time  of  exposure,  and 
stood  in  its  place  in  the  rack  as  will  be  explained  below. 


Fig.  90. — Block  for  subculture  tubes  (Anderson  and  McClintic,  in  Bulletin 
No.  82,  Hygienic  Laboratory). 


Fig.  91. — Device   for  flaming  inoculating   loops     (Anderson    and   McClintic, 
in  Bulletin  No.  82,  Hygienic  Laboratory). 

The  transplantations  from  the  seeding  tubes  to  the  culture  tubes 
are  to  be  made  every  2%  minutes  up  to  15  minutes,  so  that  for  each 
strength  of  dilution  to  be  tested,  there  will  be  six  tubes.  In  addition 
to  these  test-tubes  there  will  be  four  dilutions  of  phenol  to  act  as 
controls  so  that  every  2%  minutes  ten  transplantations  must  be 
made.  As  2%  minutes  contain  150  seconds,  and  as  the  picking  up 
and  opening  of  the  subculture  tube,  the  transfer  of  the  seed-culture 
to  the  medium,  the  replacement  of  the  stopper  and  the  return  of  the 
tube  to  the  rack  require  about  15  seconds  at  the  hands  of  an 


258  Value  of  Antiseptics 

expert    manipulator,    the    ten    tubes   in    the    series    comprise   the 
maximum  number  that  can  be  handled. 

The  illustration  shows  one  of  the  racks,  and  indicates  how  the 
tubes  are  placed  in  ten  rows  of  six  each,  each  row  with  an  empty 
hole  on  the  left.  As  the  first  tube  of  each  series  is  inoculated,  it 
is  stood  in  the  left-hand  empty  hole,  the  second  stood  in  the  hole 
from  which  the  first  was  taken,  the  third  in  that  from  which  the 
second  was  taken,  and  so  on,  so  that  there  is  always  an  empty 
hole  to  show  the  operator  which  tube  to  take  up  for  the  next 
inoculation. 

The  Technic  of  Determining  the  Phenol  Coefficient. — Everything 
being  ready  as  outlined  above,  one  proceeds  as  follows:  The 
24-hour  bouillon  culture  of  B.  typhosus  is  shaken,  then  poured  through 
a  sterile  filter-paper  in  a  sterile  glass  funnel  and  caught  in  a  sterile 
tube.  In  this  way  clumps  of  bacteria  are  removed  and  uniformly 
distributed  bacteria  secured  for  addition  to  the  "seeding  tubes." 

Exactly  5  cc.  of  each  dilution  to  be  tested  is  now  measured  into 
a  seeding  tube.  To  economize  glassware  the  same  pipet  may  be 
used  for  a  whole  series,  by  beginning  at  the  lowest  dilution,  meas- 
uring out  the  necessary  5  cc.  into  the  first  seeding  tube,  with  a 
5-cc.  delivery  pipet.  The  contents  of  the  pipet  are  then  thor- 
oughly blown  out,  and  a  pipetf ul  of  the  next  weaker  dilution  taken 
up  to  wash  out  the  pipet.  After  this  has  been  thoroughly  blown 
out  and  thrown  away,  a  pipetful  of  this  second  strength  of 
diluted  disinfectant  is  carefully  measured  into  a  second  seeding 
tube,  after  which  the  same  is  done  with  each  remaining  dilution 
in  turn.  The  tubes  are  so  marked  and  .so  arranged  in  the  rack  of 
the  -water-bath  that  no  mistake  can  be  made  in  transplanting  from 
them  in  regular  order  later.  As  each  tube  is  filled,  the  stopper  is 
replaced  and  when  all  have  been  filled  and  stood  in  the  rack,  it  is 
placed  in  the  water-bath  and  the  temperature  raised  to  2o°C. 
Anderson  and  McClintic  do  not  use  cotton  plugs  for  the  seeding 
tubes  but  sterilize  them,  open  end  down  in  a  paper-lined  wire 
basket.  Some  feel  safer,  however,  in  using  tubes  with  plugs.  The 
culture  now  being  filtered,  and  the  seeding  tubes  each  with  the  re- 
quired 5  cc.  of  each  dilution  of  the  disinfectant  to  be  tested,  all 
at  2o°C.  in  the  water-bath,  the  subculture  tubes  marked  and 
stood  in  their  respective  places  in  the  racks,  sterilized  pipets  at 
hand,  and  four  or  six  platinum  loops  on  the  block  ready  sterilized, 
with  the  burner  in  place  ready  to  re-sterilize  them,  the  technic  is 
continued  by  the  addition  of  the  culture  to  the  seeding  tubes.  At 
this  point  one  should  make  a  slight  calculation:  if  the  culture  is 
to  be  added  to  each  of  ten  of  the  seeding  tubes,  it  must  be  done 
before  the  expiration  of  150  seconds  or  2^  minutes  for  at  the  con- 
clusion of  that  time,  the  first  transplantation  from  each  seeding 
tube  to-  a  culture  tube  must  take  place.  We  have  averaged  15 
seconds  for  each  operation.  If  each  transfer  takes  an  average  of 


Testing  Germicidal  Value  of  Liquids  259 

15  seconds,  the  operator  must  have  every  detail  of  the  technic 
so  well  in  hand,  and  the  materials  so  conveniently  placed,  etc., 
that  he  can  complete  the  entire  performance  of  the  technic  from 
the  addition  of  the  culture  to  the  seeding  tubes  to  the  last  trans- 
plantation from  seeding  tubes  to  subculture  tube  without  a  hesita- 
tion and  without  a  distraction.  It  is  on  account  of  the  necessity  of 
this  "continuous  performance"  that  such  care  was  taken  to  point 
out  the  exact  details  of  apparatus  and  materials  needed,  before 
describing  the  technic. 

To  return  to  the  seeding  of  the  tubes,  a  sterile  pipet  graduated 
in  Y\  o  cc-  is  used.  The  cotton  stoppers  are  removed  from  the 
seeding  tubes  and  thrown  away  as  of  no  further  use.  One  by 
one  as  the  time  arrives,  tubes  are  taken  in  one  hand,  inclined  to 
an  angle  of  about  45  degrees,  while  the  tips  of  the  pipet  are  lightly 
touched  to  that  side  of  the  tube  from  which  the  fluid  has  run 
away  on  account  of  the  slanting,  and  exactly  o.i  cc.  of  the  cul- 
ture delivered.  This  may  under  no  circumstances  take  longer 
to  perform  than  15  seconds,  and  if  one  succeed  in  finishing  it 
in  a  shorter  time,  he  must  wait  until  the  calculated  time  arrives 
before  delivering  the  culture  into  the  next  tube  and  so  on  until 
the  end  is  reached.  Each  tube  is  given  three  gentle  shakes  after 
being  straightened  up,  then  returned  to  the  water-bath. 

With  a  ten-tube  series,  and  a  time  allowance  of  15  seconds  for 
each  tube,  the  entire  series  of  tubes  is  no  sooner  completed  than 
the  time  (2^  minutes)  for  making  the  first  series  of  transplanta- 
tions to  the  subculture  tubes  has  arrived.  The  operator  therefore 
seizes  at  once  the  first  of  the  culture  tubes  in  the  2j^-minute  series 
with  one  hand,  and  a  sterile  platinum  loop  with  the  other.  He 
cautiously  removes  the  cotton  plug  from  the  culture  tube,  and 
at  the  proper  moment  introduces  the  platinum  loop  into  the  first 
seeding  tube  all  the  way  to  its  bottom,  withdraws  it,  and  carries 
one  drop  of  the  contained  fluid  into  the  first  subculture  tube  which 
he  plugs  and  places  in  the  empty  hole  to  the  left  of  the  row  in  the 
block,  at  once  taking  up  its  neighbor  on  the  right.  As  only  15 
seconds  are  allowed  for  each  such  transfer,  the  operator  must  pro- 
ceed without  hesitation.  There  is  no  time  to  sterilize  the  platinum 
loop,  so  he  lays  it  on  the  block,  pushes  the  flame  under  it  and  takes 
up  an  already  sterilized  loop  with  which  he  performs  the  same  act 
of  transplantation  for  the  second  tube  that  was  done  for  the  first, 
doing  it  on  the  appropriate  second  of  time,  and  so  continuing 
through  the  whole  series. 

Every  test  of  the  phenol  coefficient  of  disinfection  must  em- 
brace two  such  series,  one  made  with  the  dilutions  of  the  phenol 
that  is  to  act  as  the  standard,  the  other  made  with  the  dilutions  of 
the  disinfectant  to  be  determined.  If,  however,  a  variety  of  dif- 
ferent germicides  are  to  be  tested  the  same  day,  one  phenol  test 
will  answer  the  requirement  of  the  whole  group.  The  following 


260 


Value  of  Antiseptics 


tabulation  will  make  clear  the  details  of  a  test   (Table   17  from 
Anderson  and  McClintic's  paper). 

TABLE  17 
Name  "A." 

Temperature  of  medication  2o°C. 

Culture  used.     B.  typhosus,  24-hour  extract  broth-filtered. 
Proportions  of  culture  and  disinfectant,  o.i  cc.  X  5  cc. 


Sample 

Dilution 

Time  culture  exposed  to  action  of  disin- 
fectant for  minutes 

Phenol 
coefficient 

2y2 

5 

7K 

10 

I21^ 

15 

Phenol 

1:80 

1:90 

:  100 

I 

- 

375X650 
80    no 

- 

2 

Disinfectant, 

"A" 

:  no 

'350 

'375 

1400 

'425 

I 

: 

~ 

..+.. 

+ 

4.69X5.91 

2 

5-3° 

- 

- 

- 

1500 

+ 

+ 

— 

— 

— 

— 

'550 
:6oo 

| 

| 

| 

+ 

I 

I 

1650 

+ 

+ 

+ 

+ 

+ 

— 

1700 

+ 

+ 

+ 

+ 

+ 

+ 

To  calculate  the  phenol  coefficient,  the  figure  representing  the 
degree  of  dilution  of  the  weakest  strength  of  the  disinfectant  that 
kills  within  2^  minutes  is  divided  by  the  figure  representing  the 
degree  of  dilution  of  the  weakest  strength  of  the  phenol  control 
that  kills  in  the  same  time.  The  same  is  done  for  the  weakest 
strength  that  kills  in  15  minutes.  The  mean  of  the  two  is  the 
coefficient.  The  coefficient  of  any  disinfectant  may,  for  practi- 
cal purposes,  be  defined  as  the  figure  that  represents  the  ratio  of 
the  germicidal  power  of  the  disinfectant  to  the  germicidal  power  of 
the  phenol,  both  having  been  tested  under  the  same  conditions. 

As  many  disinfectants  and  germicides  are  greatly  modified  through 
precipitation,  combination  or  other  transformation  in  the  presence 
of  organic  matter,  in  all  of  those  whose  coefficient  is  considerably 
more  than  i,  it  is  wise  to  perform  a  second  series  of  tests  in  which 
the  disinfectant  is  tested,  and  the  control  tests  made  in  the  presence 
of  organic  matter  and  the  coefficient  calculated  accordingly.  It  is 
usually  found  that  under  these  conditions  the  coefficient  falls.  In 
a  general  way,  those  disinfectants  are  most  valuable  for  general 
employment,  whose  coefficients  are  highest  in  the  presence  of 
organic  matter  in  the  test  solutions. 

The  difference  in  the  details  of  the  test  given  and  the  new  test  to 
be  made  are  as  follows: 

i.  The  test  dilutions  are  made  20  per  cent,  stronger  to  allow  for 
the  dilution  made  by  the  addition  of  the  solution  of  organic  matter. 


Testing  Germicidal  Value  of  Liquids  261 

2.  An  organic  matter  solution  is  to  be  prepared.  It  consists  of 
water  containing  10  per  cent,  of  peptone  and  5  per  cent,  of  gelatin. 
The  solids  are  dissolved  and  the  solution  sterilized.  Titration  is 
not  essential. 

The  variations  in  technic  are  simple.  Of  the  dilutions  made 
20  per  cent,  stronger  than  for  the  other  experiment,  4  cc.  (not  5  cc.) 
are  measured  into  each  seeding  tube.  The  culture  after  being  filtered 
is  added  to  the  organic  matter  in  the  proportion  of  o.i  cc.  to  each 
i  cc.  to  be  employed  in  seeding.  The  addition  of  i.i  cc.  of  the 
organic  solution  culture  mixture  to  each  seeding  tube,  gives  a  total 
of  5  cc.  of  diluted  disinfectant  containing  o.i  cc.  of  culture  and  a 
total  of  2  per  cent,  of  peptone  and  i  per  cent,  of  gelatin.  Except 
for  the  slight  difference  in  the  dilutions  and  the  seeding  with  mixed 
culture  and  organic  fluid  the  method  is  the  same,  and  the  method 
of  calculating  the  results  is  the  same. 

Anderson  and  McClintic  point  out  that  it  is  manifestly  cheaper 
to  purchase  a  disinfectant  for  60  cents  a  gallon  than  to  purchase  one 
for  30  cents  a  gallon,  providing  the  former  has  four  times  the  efficiency 
of  the  latter.  The  true  cost  of  a  disinfectant  can  only  be  deter- 
mined by  taking  into  consideration  the  phenol  coefficient  and  the 
cost  of  the  disinfectant  per  gallon.  The  cost  of  a  disinfectant 
per  100  units  of  efficiency  as  compared  with  pure  phenol  is  obtained 
by  first  dividing  the  cost  per  gallon  of  pure  phenol;  the  efficiency 
ratio  is  of  course  obtained  by  dividing  the  coefficient  of  the  dis- 
infectant by  the  coefficient  of  phenol,  but  as  the  coefficient  is  al- 
ways i,  the  efficiency  ratio  is  represented  by  the  phenol  coefficient 
of  the  disinfectant. 

The  cost  ratio  divided  by  the  efficiency  ratio  (the  coefficient  of 
the  disinfectant)  gives  the  cost  of  the  disinfectant  per  unit  of 
efficiency  as  compared  with  the  cost  per  unit  of  efficiency  of  pure 
phenol  =  i.  By  multiplying  by  100  the  relative  cost  of  100  units 
is  obtained  thus: 

Cost  of  disinfectant    .  Coefficient  of  disinfec- 

per  gallon.  _  ,  .  .  tant.  _  (  =  Efficiency 

Cost  of  phenol    per  "    Coefficient  of  phenol,      ratio.) 

gallon.  (  =  I-) 

=  cost  of  the  disinfectant  per  unit  of  efficiency  as  compared  with 
phenol  =  i,  and  by  multiplying  by  100  the  cost  of  100  units  is 
obtained.  For  instance,  the  cost  of  disinfectant  "Can"  is  $0.30 
per  gallon  and  it  has  a  coefficient  of  2.12;  the  cost  of  phenol  is  $2.67 
and  it  has  a  coefficient  of  i.  Then, 


2.12 

---  =  0.052 
2.67         i 


Therefore,  the  comparative  cost  per  unit  of  efficiency  of  "Can" 
and  phenol  respectively,  is  as  0.052  :  i;  or,  by  multiplying  by  100, 
the  relative  cost  per  100  units  —  5.2  :  100  is  obtained. 


262  Value  of  Antiseptics 

Gaseous  Disinfection. — If  the  germicide  to  be  studied  be  a  gas, 
as  in  the  case  of  sulphurous  acid  or  formaldehyd,  a  different  method 
must,  of  course,  be  adopted. 

It  may  be  sufficient  to  place  a  few  test-tube  cultures  of  various 
bacteria,  some  with  plugs  in,  some  with  plugs  out,  in  a  closed 
chamber  in  which  the  gas  is  evolved.  The  germicidal  action  is 
shown  by  the  failure  of  the  cultures  to  grow  upon  transplantation 
to  fresh  culture-media.  This  crude  method  may  be  supplemented 
by  an  examination  of  the  dust  of  the  room.  Pledgets  of  sterile 
cotton  are  rubbed  upon  the  floor,  washboard,  or  any  dust-collecting 
surface  present,  and  subsequently  dropped  into  culture  media. 
Failure  of  growth  under  such  circumstances  is  very  certain  evidence 
of  good  disinfection.  These  tests  are,  however,  very  severe,  for  in 
the  cultures  there  are  immense  numbers  of  bacteria  in  the  deeper 
portions  of  the  bacterial  mass  upon  which  the  gas  has  no  oppor- 
tunity to  act,  and  in  the  dust  there  are  many  sporogenous  organisms 
of  extreme  resisting  power.  Failure  to  kill  all  the  germs  exposed  in 
such  manner  is  no  indication  that  the  vapor  cannot  destroy  all 
ordinary  pathogenic  organisms. 

A  more  refined  method  of  making  the  tests  consists  in  saturating 
strips  of  blotting-paper,  absorbent  cotton,  various  fabrics,  etc., 
with  cultures  and  exposing  them,  moist  or  dry,  to  the  action  of  the 
gas.  Such  materials  are  best  made  ready  in  Petri  dishes,  which  are 
opened  immediately  before  and  closed  immediately  after  the  ex- 
periment. If,  when  transferred  to  fresh  culture  media,  the  ex- 
posed objects  fail  to  give  any  growth,  the  disinfection  has  been 
thorough  so  far  as  the  particular  test  organism  is  concerned.  If  the 
penetrating  power  of  a  gas,  such  as  formaldehyd,  is  to  be  tested,  it 
can  be  done  by  inclosing  the  infected  paper  or  fabrics  in  envelopes, 
boxes  perforated  with  small  holes,  tightly  closed  pasteboard  boxes, 
and  by  wrapping  them  in  towels,  blankets,  mattresses,  etc. 

Easier  of  execution,  but  rather  more  severe,  is  a  method  in 
which  cover-glasses  are  employed.  A  number  of  them  are  sterilized, 
spread  with  cultures  of  various  bacteria,  allowed  to  dry,  and  then 
exposed  to  the  gas  as  long  as  required.  They  are  subsequently 
dropped  into  culture  media  to  permit  the  growth  of  the  organisms 
not  destroyed. 

Animal  experiments  may  also  be  employed  to  determine  whether 
or  not  a  germ  that  has  survived  exposure  to  the  action  of  reagents 
has  its  pathogenic  power  destroyed.  An  excellent  example  of  this 
is  seen  in  the  case  of  the  anthrax  bacillus,  a  virulent  form  of  which 
will  kill  rabbits,  but  after  being  grown  in  media  containing  an  in- 
sufficient amount  of  a  germicide  to  kill  it,  will  often  lose  its  rabbit- 
killing  power,  though  still  able  to  fatally  infect  guinea-pigs,  or  may 
lose  its  virulence  for  both  rabbits  and  guinea-pigs,  though  still 
able  to  till  white  mice. 


CHAPTER  XVIII 
BACTERIO-VACCINES 

A  BACTERIG-VACCINE  is  a  culture  of  micro-organisms  so  modified  as 
to  be  no  longer  a  source  of  dangerous  infection,  and  so  administered 
as  to  stimulate  the  body'  defenses  and  thus  assist  either  in  pre- 
venting or  overcoming  more  virulent  infection. 

The  small  amount  of  benefit  that  occurred  from  the  employ- 
ment of  the  Oriental  method  of  "inoculating  against  small-pox" 
was  based  upon  the  theory  that  virus  of  low  virulence,  obtained 
from  a  sporadic  case  of  small-pox  if  introduced  into  the  healthy 
body,  must  result  in  a  mild  attack  of  the  disease,  by  which  the 
individual  would  be  left  immune  against  the  more  virulent  viruses 
by  which  epidemics  of  the  disease  are  brought  about.  The  observa- 
tion of  Jenner,  that  the  virus  of  cow-pox  would  protect  against 
small-pox,  led  to  the  supposition  that  the  essential  causes  of  the 
two  diseases  had  originally  been  the  same,  but  had  so  diminished 
that  the  one  became  comparatively  harmless  for  man  after  many 
generations  of  residence  in  the  cow. 

The  success  of  Pasteur's  preventive  inoculation  against  chicken- 
cholera  depended  upon  the  fact  that  the  bacilli  of  the  disease  rapidly 
lost  their  disease-producing  power  when  grown  artificially  in  cul- 
ture-media, though  they  still  retained  the  power  of  effecting  a 
change  in  the  fowls  which  thereafter  remained  immune.  His  vac- 
cination against  anthrax  was  based  upon  the  observation  that  the 
spore-forming  power  and  virulence  of  the  anthrax  bacillus  could  be 
destroyed  by  cultivation  at  temperatures  beyond  a  certain  point, 
and  that  animals  infected  with  bacilli  of  this  modified  form  subse- 
quently resisted  more  virulent  infections.  His  vaccination  against 
rabies  was  based  upon  the  supposed  diminution  in  virulence  that 
the  unknown  micro-organisms  underwent  when  exposed  to  artificial 
inspissation  of  the  nervous  tissue  in  which  they  were  contained. 
Such  organisms  of  very  low  virulence  protected  against  those  of 
higher  virulence,  and  so  on. 

From  the  periods  during  which  these  early  observations  were 
made,  to  the  present  time,  when  the  term  "  bacterio- vaccine "  is 
in  daily  use,  studies  in  immunity  have  been  conducted  in  so  great 
a  variety  of  ways  by  such  a  multitude  of  investigators,  that  it  be- 
comes tedious  to  endeavor  to  trace  the  logical  and  orderly  steps 
that  lead  to  present  knowledge,  theory  and  practice.  Two  names, 
however,  stand  out  conspicuously  in  connection  with  the  present 
topic,  because  of  the  importance  of  their  contributions,  those  of 
Haffkine  and  Wright.  The  former  used  heated  and  killed  cultures 

263 


264  Bacterio- vaccines 

of  the  cholera  spirillum  as  a  prophylactic  against  cholera,  and  later 
with  equal  success,  heated  and  killed  cultures  of  the  plague  bacillus 
as  a  prophylactic  against  plague.  Wright  somewhat  modified  the 
method,  by  using  two  or  even  three  doses  of  modified  cultures  of 
the  typhoid  bacillus  at  intervals  of  ten  or  even  twenty  days,  to  secure 
complete  prophylaxis  against  typhoid  fever. 

From  prophylactic  measures  it  was  but  a  step  to  therapeutic 
measures,  and  the  endeavor  to  facilitate  the  cure  of  disease  by  the 
administration  of  cultures  of  vaccine.  The  patient  suffering  from 
an  infectious  disease  was  already  impressed  by  the  toxic,  enzymic 
or  other  disease-producing  substances  in  his  body,  and  the  ad- 
ministration of  cultures  of  micro-organisms  seemed  like  adding 
so  much  fuel  to  an  already  widespread  conflagration.  Indeed, 
experience  and  experiment  seemed  to  prove  this  to  be  the  case,  for 
when  by  any  mischance  a  patient  in  the  early  stages  of  plague 
received  an  injection  of  the  Haffkine  plague  prophylactic,  he  straight- 
way became  much  injured  by  the  added  culture  and  might  even 
die  quickly. 

But  there  are  certain  infections  in  which  conditions  are  different 
both  with  regard  to  the  bacteria  and  the  disease.  Thus,  a  certain 
micro-organism  with  limited  power  of  invasion  and  with  difficultly 
soluble  toxic  products  (endo- toxins),  whose  injurious  effects  are 
local  and  limited  in  extent,  particularly  when  their  effects  are  pro- 
longed and  the  disturbances  chronic,  are  essentially  different  from 
actively  invasive  agents  that  quickly  over-run  the  body,  or  those 
with  considerable  soluble  products  by  which  it  is  generally  disturbed. 

In  the  former  group  it  is  not  unreasonable  to  hope  that  through  a 
method  of  treatment  by  which  the  general  body  defences  are  stimu- 
lated, the  local  infections  may  be  overcome.  Such  cases  of  dis- 
ease were,  therefore,  selected,  especially  by  Wright,  for  investi- 
gation and  treatment.  Success  of  varying  degree  has  followed, 
and  though  it  is  difficult  to  calculate  accurately  the  benefits  obtained 
in  cases  that  are  not  susceptible  of  numerical  expression,  the  almost 
uniform  opinion  of  clinical  and  laboratory  men  is  to  the  effect  that 
certain  cases  of  sluggish  infection,  with  little  tendency  to  recover  are 
benefited  and  sometimes  rapidly  cured  by  treatment  with  bacterio- 
vaccines. 

From  these  preliminary  considerations  it  should  be  clear  to  the 
reader  that  the  theoretical  conditions  necessary  to  success  are  the 
following: 

1.  That  the  disease  should  be  of  subacute  or  chronic  duration. 

2.  That  it  should  be  fairly  well  localized. 

3.  That  it  should  be  caused  by  a  micro-organism  incapable  of  ready  invasion 
or  much  soluble  toxin  formation. 

4.  That  the  micro-organism  be  known  and  capable  of  cultivation  so  that  the 
appropriate-specific  vaccine  can  be  made. 

From  these  conditions  certain  lesions  resulting  from  infection  by 
pus  cocci,  colon  bacilli,  acne  bacilli,  typhoid  bacilli  (post-typhoidal 


Method  of  Making  the  Vaccine  265 

suppurations),  tubercle  bacilli,  etc.,  etc.,  ought -to  be  appropriate. 
And,  indeed,  for  them  the  treatment  is  highly  recommended,  and  in 
many  cases  remarkable  success  is  claimed. 

Remembering  that  the  reactions  of  immunity  are  specific,  it  is 
imperative  that  the  essential  organism  of  the  lesion  be  found  and 
cultivated,  and  cultures  of  that  organism  used  in  the  treatment. 
So  important  is  this  that  Wright  insists  that  only  "autogenous 
vaccines" — that  is,  vaccines  made  of  cultures  of  bacteria  cultivated 
from  the  very  lesion  to  be  treated — be  used.  This  somewhat  limits 
the  usefulness  of  the  method  for  the  rank  and  file  of  practitioners 
can  scarcely  be  supposed  to  have  the  knowledge,  apparatus,  or  time 
required  for  carrying  out  the  technic,  nor  can  all  patients  afford 
to  patronize  the  laboratory  man.  Commercial  manufacturers  are 
therefore  justified  in  the  preparation  and  sale  of  what  are  known  as 
" stock  vaccines"  that  can  be  tried  in  lieu  of  autogenous  vaccines, 
though  in  checking  up  the  results  note  should  always  be  taken  of 
the  fact  that  " autogenous"  or  "stock"  vaccines  were  used. 

In  spite  of  the  general  principles  laid  down  above,  there  are  re- 
ports and  observations  to  show  that  the  theoretical  considerations 
may  be  faulty  and  that  in  some  cases  the  method  of  treating  by 
vaccination  may  be  beneficial  in  acute  maladies,  even  when  the 
condition  to  be  treated  is  toxic.  It  will  be  necessary,  however,  to 
secure  much  more  evidence  with  regard  to  the  employment  of  the 
method  in  such  cases  before  it  can  be  recommended  as  sound 
practice. 

Should  a  case  of  appropriate  kind,  when  investigated,  yield  more 
than  one  species  of  micro-organism,  of  such  kind  as  to  make  it  un- 
certain which  is  responsible  for  the  injury  done,  both  should  be 
cultivated,  two  vaccines  made  and  mixed,  and  both  infections 
simultaneously  antagonized. 

The  Method  of  Making  the  Vaccine. — A  pure  culture  of  the 
necessary  micro-organisms  is  obtained  from  the  lesion  to  be  treated, 
and  cultivated  in  agar-agar. 

One  pint  "Blake  bottles,"  pint  or  quart  white  glass  whisky  flasks,  or  other 
good  sized  bottles  with  large  flat  sides,  are  selected  and  washed.  Into  each 
enough  melted  agar-agar  is  filled  to  spread  out  over  one  of  the  flat  surfaces  to  a 
thickness  of  about  i  centimeter,  after  which  a  cotton  plug  is  placed  in  the 
mouth  of  the  bottle,  and  it  and  its  contents  are  sterilized  in  the  autoclave.  Upon 
removal,  after  sterilization,  the  bottle  is  laid  on  its  side  so  as  to  distribute  the 
agar-agar  and  permit  it  to  solidify  over  the  greatest  surface,  without  flowing 
into  the  neck  and  touching  the  cotton  stopper.  To  the  agar-agar  culture  of  the 
micro-organism  to  be  used,  about  10  cc.  of  sterile  0.85  per  cent,  sodium  chloride 
solution  is  added,  the  culture  mass  being  detached  with  a  platinum  loop  and 
thoroughly  mixed  with  the  fluid.  When  the  agar-agar  is  firm,  each  bottle 
receives  by  means  of  a  carefully  sterilized  pipet,  about  i  cc.  of  the  culture 
suspension  which  is  thoroughly  distributed  over  the  entire  flat  surface  of  the 
agar-agar  by  tilting  the  bottle  this  way  and  that  until  it  has  been  completely 
covered.  The  bottles  are  then  placed  in  the  incubating  oven,  lying  upon  the 
side  so  as  to  permit  the  bacteria  to  vegetate  undisturbed  upon  the  moist  flat 
surface  of  the  medium.  After  24  hours,  the  growth  having  matured,  the  bottles 
are  removed  and  about  10  cc.  of  sterile  distilled  water  containing  0.85  per  cent. 


266  Bacterio-vaccines 

of  sodium  chloride  and  0.5  per  cent,  of  phenol  is  added  to  each,  for  the  purpose 
of  washing  off  the  bacteria  that  have  grown.  This  is  done  by  tilting  the  bottle 
and  permitting  the  solution  to  wash  over  and  over  the  surface.  If  the  culture 
does  not  detach,  it  may  be  necessary  to  remove  it  with  a  sterilized  glass  rod,  or 
by  means  of  a  sterile  swab  made  by  fastening  a  small  pledget  of  cotton  batting 
upon  the  end  of  a  wire. 

When  the  growth  is  detached  and  thoroughly  mixed  with  the  salt  solution, 
it  is  removed  to  a  sterile  receptacle  by  means  of  a  sterile  pipet. 

What  is  next  done  will  depend  upon  the  theory  upon  which  the 
treatment  is  based.  The  culture  washings  contain:  (A)  sub- 
stances derived  from  the  culture-medium  that  certainly  cannot  be 
regarded  as  useful  or  beneficial  and  may  be  harmful; 

(B)  bacterial  products,  of  soluble  quality,  eliminated  from  the 
cells  during  the  life  activities,  some  of  which  may  be  useful; 

(C)  the  bacteria  themselves,  which  with  their  contained  prod- 
ucts— endo- toxins,  etc. — are  commonly  regarded  as  the  essential' 
immunizing  agents. 

If  one's  theory  is  that  the  bacterial  cells  are  essential,  and  there 
seems  to  be  a  growing  tendency  toward  this  view,  further  treatment 
is  necessary  before  actually  preparing  the  vaccine  for  administra- 
tion; if,  however,  the  collected  products  of  their  growth  are  thought 
to  be  of  partial  or  equal  value,  and  are  to  be  preserved,  this  cannot 
be  done  without  also  retaining  the  less  desirable  matters  from  the 
culture-medium. 

Let  us  suppose  that  only  the  bacterial  cells  are  to  be  employed. 

The  suspension  of  bacteria,  under  these  circumstances,  is  transferred  to 
appropriate  sterile  tubes,  plugged,  and  whirled  in  a  powerful  centrifuge  until 
the  bacteria  are  thrown  down  to  the  end  of  the  tube,  leaving  the  supernatant 
fluid  fairly  clear.  The  fluid  is  then  removed  by  decantation  or  with  a  pipet, 
and  replaced  by  an  equal  volume  of  0.5  per  cent,  phenol  in  0.85  per  cent,  sodium 
chloride  solution  in  distilled  water.  In  this  the  sediment  is  thoroughly  mixed  by 
stirring.  As  the  bacteria  are  often  in  masses,  groups  or  chains,  it  is  now  necessary 
to  separate  them.  This  is  best  done  by  adding  a  few  small  glass  beads  to  the 
contents  of  the  tubes,  changing  the  cotton  stopper  for  a  sterile  rubber  cork,  and 
shaking  either  in  a  shaking  machine  or  by  hand,  until  it  can  be  supposed  that  the 
micro-organisms  are  all  separated.  This  is  easily  accomplished  by  the  aid  of  the 
shaking  machine  but  is  tedious  to  effect  by  hand.  The  tube  is  then  returned  to 
the  centrifuge  and  again  whirled  until  the  bacteria  are  again  sedimented,  after 
which  the  fluid  is  again  removed  and  again  replaced  and  the  bacteria  again  dis- 
tributed. A  few  turns  in  the  centrifuge  now  throw  down  particles  of  culture- 
media  and  contained  flakes  of  the  culture  and  leave  a  uniformly  clouded  fluid 
above. 

If  it  be  desired  to  conserve  all  of  the  bacterial  products,  the 
washings  from  the  culture  bottles  are  immediately  transferred  to 
the  appropriate  tube,  shaken  with  the  glass  beads,  given  a  few  turns 
in  the  centrifuge  to  throw  out  flakes  of  culture  and  culture-media, 
and  we  again  arrive  at  the  point  of  having  a  uniformly  cloudy  fluid 
with  which  to  continue  the  preparation  of  the  vaccine. 

If  the  vaccine  is  to  be  of  scientific  value,  it  should  be  made  in 
such  manner  that  its  composition  represents  what  is  desired — 
bacterial  cells  only,  or  bacterial  cells  with  their  collected  products — 
and  some  means  should  be  provided  by  which  a  reasonably  accurate 


Method  of  Making  the  Vaccine  267 

determination  of  its  value  can  be  estimated.  This  is  done  by  cal- 
culating the  number  of  contained  bacteria  per  cubic  centimeter 
of  the  fluid,  and  then  either  diluting  or  concentrating  by  means  of 
centrifugation  until  an  appropriate  result  is  reached.  As  the  con- 
centration by  centrifugation  is  more  difficult  than  dilution  it  is  best 
to  take  care  at  the  very  beginning  of  the  process  not  to  add  too 
much  fluid  to  the  culture  bottles  for  the  purpose  of  washing  off  the 
culture.  Whatever  dilution  of  the  final  product  may  be  necessary 
is  made  by  the  use  of  the  o  5  per  cent,  phenol  solution. 

The  most  ready  method  of  calculating  the  number  of  bacteria 
in  the  fluid  is  that  of  A.  E.  Wright  which  will  be  found  in  the  chapter 
upon  the  "Calculation  of  the  Opsonic  Index." 

After  having  determined  the  number  of  bacterial  cells  per  cubic 
centimeter  of  fluid,  dilution  with  the  phenol  solution  is  made  until 
single  doses  are  contained  in  quantities  easily  injected  into  the 
patient.  As  the  doses  vary  with  the  particular  organism  to  be 
injected,  the  operator  must  calculate  from  the  number  of  bacteria 
in  the  fluid,  how  much  solution  must  be  added  to  constitute  a  dose. 
Several  doses  of  each  desired  size  should  be  prepared.  Quantities 
of  the  dilutions  containing  single  doses  or  a  number  of  doses  as  may 
be  preferred  are  now  transferred,  by  means  of  a  sterile  pipet,  into 
previously  sterilized,  appropriate  sized  " ampules"  or  glass  bulbs 
made  for  the  purpose,  and  the  necks  sealed  in  a  flame. 

The  bacteria  are,  however,  still  alive,  and  though  many  of  them 
no  doubt  undergo  autolysis  in  the  phenol  salt  solution,  it  is  nec- 
essary to  make  certain  that  none  remains  alive  to  infect  the  patient. 

The  destruction  of  the  vitality  of  the  micro-organisms  which  is 
the  final  step  in  the  process  of  vaccine  preparation  is  effected  by  ex- 
posure to  the  lowest  temperature  that  is  known  to  be  positively 
destructive.  As  spore-producing  micro-organisms  may  maintain 
this  vitality  at  temperatures  beyond  ioo°C.,  at  which  the  micro- 
organismal  substance  as  well  as  their  products  are  altered  by  coagula- 
tion and  other  destructive  transformation,  they  are  inappropriate 
organisms  to  employ  for  purposes  of  vaccines,  unless,  through  some 
such  ingenious  means  as  was  devised  by  Pasteur  for  the  anthrax 
bacillus,  the  production  of  spores  can  be  prevented. 

With  very  few  exceptions  non-sporogenous  bacteria  are  destroyed 
by  exposure  for  60  minutes  to  a  temperature  of  6o°C.  Should  any 
escape  destruction,  they  are  probably  so  injured  as  to  be  incapable  of 
further  injurious  effect  upon  the  human  body. 

The  destruction  of  the  bacteria  is,  then,  effected  by  heat: 

The  ampules  of  vaccine  are  placed  in  some  sufficiently  commodious  receptacle 
filled  with  water,  the  heat  being  supplied  by  a  flame  below,  and  the  temperature 
determined  by  a  thermometer  whose  bulb  is  at  the  center  of  the  bath.  When 
small  quantities  of  the  vaccine  are  to  be  made  for  special  cases,  a  large  beaker 
supported  upon  an  asbestos  plate  upon  a  chemical  tripod  and  heated  by  a 
Bunsen's  flame  answers  very  well.  The  burner  is  allowed  to  heat  the  bath  until 
the  proper  temperature  is  reached,  when  it  is  removed.  As  soon  as  the  tern- 


268  Bacterio-vaccines 

perature  begins  to  fall,  it  is  replaced.     Thus  by  alternately  heating  and  re- 
moving the  source  of  heat  for  60  minutes,  the  destruction  is  affected. 

If  there  are  many  of  the  small  ampules,  containing  different 
doses  or  different  cultures,  each  separate  lot  may  be  done  up  in  a 
piece  of  gauze,  and  labelled. 

J.  H.  Small  uses  orange-colored  "string  tags"  for  this  purpose, 
writing  upon  them  with  either  pen  or  pencil,  and  fastening  them  to 
the  gauze  packages.  In  the  water  of  the  water-bath,  the  writing 
does  not  wash  off  of  the  tag,  but  the  color  comes  out  and  gives 
the  water  an  orange  tinge.  This  is  found  to  be  of  the  greatest  use, 
for  as  one  or  more  of  the  factory-made  ampules  commonly  cracks  in 
the  water-bath,  the  color  penetrates  the  contained  fluid.  Upon 
removal  from  the  water-bath,  to  glance  at  each  ampule  will  inform 
the  observer  whether  it  is  cracked  or  not,  through  the  change  in 
the  color  of  the  contents.  The  tags,  therefore,  subserve  a  double 
purpose. 

After  heating,  one  of  the  ampules  can  be  opened  and  a  drop  of  the 
contents  transferred  to  a  tube  of  culture  to  make  sure  that  the  bacteria 
are  no  longer  alive. 

The  vaccine  is  now  ready  for  use,  but  in  what  dose  shall  it  be  ad- 
ministered? There  is  no  other  information  upon  this  subject  than 
that  which  is  derived  from  the  experience  that  certain  doses  seem  to 
accomplish  good  without  producing  ill  effects.  Thus  experience 
with  doses  at  first  selected  arbitrarily  has  led  to  a  fairly  accurate 
standard  dosage.  As  the  beginning  dose  for  most  vaccines  50-250 
millions  may  be  recommended,  to  be  increased  to  1000  millions  or 
more,  the  injections  being  given  every  4-6  days  or  as  controlled  by 
the  opsonic  index. 

The  benefit  of  the  vaccine  is  commonly  supposed  to  depend  upon 
the  stimulation  of  the  phagocytic  cells  of  the  body.  This  is  very 
probably  the  case,  but  when  the  bacterial  bodies  are  administered, 
their  dissolution  results  in  the  liberation  of  the  contained  endo- 
toxin,  and  when  the  entire  culture  is  given,  endo-toxins  and  perhaps 
exo-toxins  and  other  substances  are  also  given  so  that  the  increased 
phagocytosis  is  not  likely  to  be  the  only  effect  of  the  treatment. 

A.  E.  Wright  who  is  a  firm  believer  in  the  stimulating  influence 
upon  the  cells  seeks  to  control  the  dosage  and  estimate  the  value  of 
the  injections  by  such  study  of  phagocytic  activity,  as  is  shown  in 
the  next  chapter.  If  after  an  injection  of  vaccine,  the  phagocytic 
activity  of  the  leukocytes  is  diminished  (negative  phase)  harm  is 
supposed  to  have  been  done  and  the  inference  is  drawn  that  the  dose 
was  too  large;  if,  on  the  other  hand,  the  phagocytic  activity  is 
increased  for  the  respective  organism,  good  is  supposed  to  have  been 
done,  and  at  the  next  injection  the  same  or  a  larger  dose  may  be 
given. 

Besredka  and  Metschnikoff  *  have  modified  the  vaccines  by  what 
*  Ann.  d.  1'Inst.  Pasteur,  1913,  xxvn,  597. 


Method  of  Making  the  Vaccine  269 

is  called  sensitization.  This  they  accomplish  by  treating  the 
bacteria  to  be  used  with  an  antiserum,  prepared  by  injecting 
animals  with  such  organisms  as  form  the  vaccine.  In  this  manner 
the  specific  bacteriolytic  amboceptors  are  supposed  to  anchor 
themselves  to  the  bacterial  cells,  and  so  pave  the  way  for 
immediate  destructive  treatment  in  the  body.  To  achieve  such 
sensitization,  some  of  the  appropriate  serum  is  added  to  the 
bacterial  suspension  which  need  not  be  subsequently  killed,  as 
the  sensitized  bacteria  meet  with  prompt  destruction  through 
the  normal  complement  of  the  body  juices.  However,  if  the 
bacteria  are  first  killed  by  heat  and  then  sensitized,  a  similar 
result  may  be  brought  about,  and  one  is  relieved  of  all  anxiety  as  to 
the  possibility  of  infection  accidentally  resulting  from  the  injections. 


CHAPTER  XIX 

THE  PHAGOCYTIC  POWER  OF  THE  BLOOD  AND  THE 
OPSONIC  INDEX 

FROM  the  time  that  Metschnikoff  connected  the  phenomena 
of  phagocytosis  with  those  of  immunity,  there  was  no  recognized 
technic  for  the  observation  and  comparison  of  the  bacteria-con- 
suming and  bacteria-destroying  power  of  the  cells  until  iqo2,  when 
Leishman*  suggested  the  following  simple  method: 

A  thin  suspension  of  bacteria  in  normal  salt  solution  is  mixed 
with  an  equal  volume  of  blood  by  drawing  in  and  out  of  a  capillary 
tube,  then  dropped  upon  a  clean  slide,  covered  carefully,  placed  in  a 
moist  chamber,  and  incubated  at  37°C.  for  a  half  hour.  The  cover 
is  then  slipped  off  carefully,  as  in  making  blood-spreads,  dried, 
stained,  and  the  number  of  bacteria  in  each  of  20  leukocytes  counted 
and  averaged.  For  comparison  with  the  normal,  the  patient's 
blood  and  normal  blood  are  simultaneously  examined. 

This  was  greatly  improved  by  Wright  and  Douglas,  f  the  accuracy 
of  whose  methods  enabled  them  to  discover  the  "opsonins,"  work 
out  the  "opsonic  index,"  and  formulate  methods  by  which  sufficiently 
accurate  observations  could  be  made  for  controlling  the  specific 
treatment  of  infectious  diseases. 

The  opsonic  theory  teaches  that  the  leukocytes  are  disinclined 
to  take  up  bacteria  unless  they  are  prepared  for  consumption  or 
phagocytosis  by  contact  with  certain  substances  in  the  serum  that 
in  some  manner  modify  them.  This  modifying  substance  is  the 
opsonin  (opsono,  I  cater  to,  I  prepare  for). 

To  make  a  test  of  the  opsonic  value  of  the  blood  it  is  necessary 
to  prepare  the  following: 

A  uniform  suspension  of  bacteria. 

A  suspension  of  washed  leukocytes  in-  physiological  salt  solution. 

The  serum  to  be  tested. 

A  normal  serum  for  comparison. 

The  Bacterial  Suspension. — This  is  prepared  like  the  similar 
suspensions  used  for  determining  agglutination,  but  with  greater 
care,  since  the  bacteria  taken  up  by  the  corpuscles  are  to  be  counted, 
and  any  variation  in  the  number  of  bacteria  with  which  they  come 
into  contact  may  modify  the  count.  It  is  also  necessary  to  avoid 
all  clumps  of  bacteria  for  the  same  reason. 

The  culture  is  best  grown  upon  agar-agar  for  twelve  to  twenty- 
four  hours,  the  bacteria  in  young  cultures  being  more  easy  to  sepa- 

*  "British  Medical  Journal,"  Jan.  n,  1002,  i,  p.  73. 
.  Royal  Soc.  of  London,"  1904,  LXXXII,  p.  357. 
270 


The  Bacterial  Suspension 


271 


rate  than  those  in  old  cultures.  Such  a  culture  may  be  taken  up 
in  a  platinum  loop,  transferred  to  a  test-tube  containing  some 
0.85  per  cent,  sodium  chloride  solution,  and  gently  rubbed  upon  the 
glass  just  above  the  fluid,  allowing  the  moistened  and  mixed  bacterial 
mass  to  enter  the  fluid  little  by  little. 
If  the  culture  be  older  or  of  a  nature  that  will  not  separate  in 


• 


Fig.  92. — Grinding  bacteria  (Miller). 

this  manner  (tubercle  bacillus),  it  may  be  necessary  to  rub  it  between 
two  glass  plates,  or  in  a  small  agate  mortar  with  a  drop  or  two  of 
salt  solution,  other  drops  being  added  one  at  a  time,  until  a  homo- 
geneous suspension  is  secured.  Such  clumps  of  bacteria  as  may 
remain  in  the  suspension  are  easily  removed  by  whirling  for  a  few 
seconds  in  a  centrifuge. 

The  next  step  is  the  standardization  of  the  suspension.  Wright 
recommends  for  this  purpose  and  for 
the  standardization  of  the  bacterio- 
vaccines  that  the  number  of  bac- 
teria shall  actually  be  counted.  This 
he  does  by  mixing  one  part  of  the 
bacterial  suspension  with  an  equal 
volume  of  normal  blood  and  three 
volumes  of  physiological  salt  solu- 
tion. After  thorough  mixing  a  smear 
is  made  upon  a  slide,  the  smear 
stained,  and  the  number  of  bacteria 
and  corpuscles  in  successive  fields  of 
the  microscope  counted  until  at  least  200  red  blood-corpuscles  have 
been  enumerated.  As  the  number  of  red  corpuscles  per  cubic  milli- 
meter of  blood  is  5,000,000,  the  number  of  bacteria  per  cubic  centi- 
meter can  be  determined  from  the  results  of  the  counting  by  a 
simple  arithmetical  process.  To  facilitate  the  counting  the  eye- 
piece of  the  microscope  is  prepared  by  the  introduction  of  a  dia- 
phragm. The  prepared  suspension  must  usually  be  greatly  diluted 
before  using,  but  the  reduction  of  bacteria  is,  of  course,  easily  cal- 


Fig.  93.— Diaphragm  of  eye- 
piece showing  hairs  in  position 
(Miller). 


272 


The  Phagocytic  Power  of  the  Blood 


culated.  It  requires  experience  to  determine  the  appropriate  number 
of  bacteria  to  be  employed.  When  this  is  once  determined,  future 
manipulations  are  made  easy,  because  one  first  makes  his  suspension, 


Fig.  94. — Photomicrograph  showing  cross-hairs,  bacteria,  and  red  blood- 
corpuscles  (Miller). 

then  enumerates  the  bacteria,  and  having  determined  their  number, 
immediately  arrives  at  the  appropriate  concentration  by  dilution. 


Fig.  95. — Collecting  blood  for  corpuscles  (Miller). 

The  Washed  Leukocytes. — It  is  not  necessary  to  have  the  leuko- 
cytes free  from  admixture  with  the  erythrocytes,  but  it  is  necessary 
to  have  large  numbers  of  them.  They  are  collected  by  citrating  the 
blood  so  as  to  prevent  coagulation,  and  then  separating  the  citrated 
plasma  from  the  corpuscles  by  centrifugalization. 


The  Washed  Leukocytes 


273 


The  hands  of  the  patient  are  washed,  and  a  piece  of  elastic  rubber 
tubing  or  some  other  convenient  fillet  wound  about  the  thumb  or 
a  finger  to  produce  venous  congestion.  With  a  convenient  lancet 
(Wright  uses  a  pricker  made  by  drawing  a  bit 
of  glass  tubing  or  a  glass  rod  to  a  fine  point  in 
the  flame)  a  prick  is  made  about  a  quarter  inch 
from  the  root  of  the  nail.  From  this  the  blood 
is  permitted  to  flow  into  small  test-tubes  pre- 
viously filled  about  three-fourths  with  1.5  per 
cent,  sodium  citrate  solution.  The  blood  and 
citrate  solution  are  mixed,  and  the  tubes  placed 
in  a  centrifuge,  balanced,  and  centrifugalized 
until  the  corpuscles  are  collected  at  the  bottom 
of  the  tube.  The  citrated  plasma  is  now  with- 
drawn and  replaced  with  0.85  per  cent,  sodium 
chloride  solution,  through  which  the  corpuscles 
are  distributed  by  shaking.  The  tubes  are  now 
again  centrifugalized  until  the  corpuscles  are 
collected,  when  the  saline  is  removed  carefully, 
the  last  drop  from  the  back  of  the  meniscus. 
In  the  corpuscular  mass  that  remains  the  leuko- 
cytes form  a  thin  creamy  layer  on  the  top. 

The  serum  to  be  tested  and  the  normal  serum  for  comparison 
are  secured  in  the  same  manner,  the  former  from  the  patient,  the 
latter  from  the  operator.  As  it  is  advisable  to  wound  the  patient 


Fig.  96. — Tube  of 
blood  and  citrate 
solution  before  and 
after  centrif  ugaliz- 
ing  (Miller). 


Fig.  97. — Removing  last  drops  of  saline  solution  (Miller). 

but  once,  the  tube  for  obtaining  the  serum  should  be  filled  at  the 
same  time  that  the  citrated  blood  is  taken. 

The  blood  to  furnish  the  serum  is  taken  in  a  small  bent  tube  shown 
in  the  illustration. 

18 


274 


The  Phagocytic  Power  of  the  Blood 


The  blood  from  the  puncture  is  allowed  to  flow  into  the  bent 
end  of  the  tube,  into  which  it  enters  by  capillary  attraction  and 
from  which  it  descends  to  the  body  of  the  tube  by  gravity.  At 
least  i  cc.  of  the  blood  is  required  to 
furnish  the  serum.  The  ends  of  the 
tube  are  closed  in  the  flame  and  the 
tube  stood  in  the  thermostat  for  fifteen 
to  thirty  minutes.  Coagulation  takes 
place  almost  immediately,  and  the  serum 
usually  separates  quickly.  If  it  does  not 
do  so,  Wright  recommends  hanging  the 
curved  arm  of  the  tube  over  the  cen- 


Fig.  98.— Special  blood  pipetet  (Miller). 

trifuge  tube  and  whirling  it  for  a  mo- 
ment or  two,  when  the  clot  is  driven 
into  the  straight  arm  of  the  tube  and 
the  clear  serum  appears  above.  The 
tube  is  then  cut  with  a  file  so  that  the 
serum  can  be  removed  when  needed. 
Mixing  the  factors  concerned  in  the  test 
is  a  matter  that  requires  practice  and 
a  steady  hand.  It  is  best  done,  as  rec- 
ommended by  Wright,  in  a  capillary  tube  controlled  by  a  rubber 
bulb.  The  object  of  the  experimenter  is  to  take  up  into  this  pi- 
pette equal  quantities  of  the  creamy  layer  of  blood-corpuscles,  of 


Fig.  99. — Opsonizing  pipette 
containing  blood-corpuscles, 
bacterial  emulsion,  and  blood- 
serum  (Miller). 


The  Washed  Leukocytes 


275 


the  blood-serum,  and  of  the  bacterial  suspension.  Wright  first 
makes  a  mark  with  a  wax  pencil  about  i  centimeter  from  the  end 
of  the  capillary  tube.  He  first  draws  up  the  leukocytic  layer  of 
blood-corpuscles  to  this  mark,  then  removing  the  tube,  permits  the 
column  to  ascend  a  short  distance.  Next  he  draws  up  the  bac- 
terial suspension  to  the  same  point,  withdraws  the  tube,  and  per- 
mits the  column  to  ascend;  then  draws  up  the  serum  to  be  taken 
to  the  same  point;  thus  in  the  same  capillary  tube  he  has  three 
equal  volumes  of  three  different  fluids,  separated  by  bubbles  of 
air.  It  is  next  necessary  to  mix  these,  which  is  done  by  repeat- 
edly expelling  them  upon  a  clean  glass  slide,  and  redrawing 
them  into  the  tube.  After  thus  being  thoroughly  mixed,  the  fluid 
is  once  more  permitted  to  enter  the  capillary  tube  and  come  to  rest 


Fig.  100. — Mixing  liquids  by  repeatedly  expelling  on  to  slide  and  redrawing 
into  pipette  (Miller). 

there.  The  end  is  now  sealed  in  a  flame,  the  rubber  bulb  removed 
and  the  tube  placed  in  a  thermostat,  or  in  case  much  work  of  the 
kind  is  being  done,  to  an  opsonizing  incubator  in  which  the  tempera- 
ture is  not  modified  by  opening  and  closing  the  doors.  The  tube 
remains  in  the  incubating  apparatus  at  37°C.  for  fifteen  minutes 
(some  use  twenty,  some  thirty,  minutes  as  their  standard),  is  then 
removed,  whirled  about  its  long  axis  between  the  thumbs  and  fingers 
a  few  times  to  mix  the  contents  from  which  the  corpuscles  have 
sedimented,  its  end  is  broken  off,  and  a  good-sized  drop  is  allowed  to 
escape  upon  a  perfectly  clean  glass  slide  and  spread  over  its  surface. 
The  spreading  is  a  matter  of  some  importance,  as  an  even  dis- 
tribution of  the  leukocytes  is  desirable.  The  capillary  tube  from 
which  the  drop  has  escaped  will  form  a  good  spreader  if  laid  flat 
upon  the  glass  and  drawn  along,  but  the  edge  of  another  slide  is 
better,  and  in  distributing  the  fluid,  it  is  better  to  push  than  to  pull 
it  with  the  end  of  the  slide,  rather  than  its  side. 


276 


The  Phagocytic  Power  of  the  Blood 


Miller*  says  that  "a  good  smear  should  be  uniform  in  consistency 
and  most  of  the  leukocytes  should  be  found  along  the  edges  and 
at  the  end.  For  convenience  in  counting,  it  is  well  to  have  the 
smear  terminate  abruptly  and  not  be  drawn  out  into  threads  or 
irregular  forms." 


Fig.  101. — A  small  incubator  of  special  design  for  opsonic  work  (Miller). 

This  mixing,  incubating,  and  spreading  is  done  twice — once 
with  the  serum  of  the  patient,  and  once  with  the  normal  serum  of 
the  operator.  The  technic  is  the  same  each  time.  In  order  that 

the  enumeration  of  the  bacteria 
taken  up  by  the  leukocytes  can 
be  accomplished,  it  is  next  neces- 
sary to  stain  the  blood  smears. 
This  can  be  done  by  any  method 
ttiat  will  demonstrate  both  the 
bacteria  and  the  cells.  For 
Fig.  102.— The  smear  (Miller).  staphylococci  and  similar  organ- 
isms, Leishman's  stain,  Jenner's 

stain,  or  J.  H.  Wright's  stains  are  appropriate.  Marino's  stain, 
recommended  by  Levaditi,f  gives  beautiful  results.  For  the 
tubercle  bacillus  the  spreads  may  be  stained  with  carbol-fuchsin 

*  "Therapeutic  Gazette,"  March  15,  1907. 

t  "Ann.  de  ITnst.  Pasteur,"  1904,  xvm,  p.  761. 


The  Washed  Leukocytes  277 

and  counterstained  with  methylene-blue,  or  perhaps  better  with 
gentian  violet  and  counterstained  with  Bismarck  brown  or  vesuvin. 

The  final  step  in  the  process  is  the  enumeration  of  the  bacteria 
in  the  corpuscles  by  averaging  the  number  taken  up  by  the  cells. 
Only  typical  polymorphonuclear  cells  should  be  selected  for  staph- 
ylococcic  cases,  and  separate  averages  made  for  polymorphonu- 
clear and  mononuclear  cells  in  tubercle  bacillus  cases.  It  is  best  to 
follow  certain  routine  methods  of  enumeration.  Some  who  content 
themselves  with  a  count  of  the  number  of  bacteria  in  20  cells, 
secure  less  accurate  results  than  those  who  count  50  cells.  It  is 
usually  best  to  count  one-third  of  the  cells  in  the  central  portion 
of  the  spread,  one-third  at  the  edge,  and  one-third  at  the  end. 
In  each  portion  no  other  selection  of  cells  should  be  made  than  the 
elimination  of  other  than  polymorphonuclear  cells  and  the  elimina- 
tion of  all  crushed  or  injured  cells;  the  others  should  be  taken  one 
after  the  other,  as  they  are  brought  into  the  field  with  the  mechanical 
stage.  After  the  bacteria  included  in  each  of  the  accepted  number 
of  cells  selected  as  the  standard  has  been  enumerated,  an  average 
is  struck. 

The  "opsonic  index"  is  determined  by  dividing  the  average 
number  in  the  patient's  serum  preparation  by  the  average  in  the 
normal  serum  preparation. 

Leishman's*  studies  of  the  phagocytic  power  of  the  blood  show 
that  in  cases  of  furunculosis,  etc.,  with  each  recrudescence  of  boils, 
there  is  a  marked  diminution  of  the  phagocytic  power  of  the  blood, 
and  with  each  improvement,  a  marked  increase. 

McFarland  and  TEnglef  found  by  an  examination  of  the  blood 
of  24  supposedly  healthy  students  and  laboratory  workers  that  it 
was  possible  to  prejudge,  by  the  phagocytic  activity  of  the  cells, 
the  past  occurrence  of  suppuration  and  present  liability  to  it. 

Wright  and  Douglas  use  the  opsonic  index  as  a  guide  to  the 
specific  therapy  of  the  infectious  diseases.  If  the  opsonic  index  is 
low  they  believe  bacterio-vaccination  is  indicated.  In  its  admin- 
istration, however,  care  must  be  taken  to  administer  a  counted 
number  of  bacteria,  and  to  make  frequent  opsonic  estimations  to 
determine  the  good  or  ill  effects  accomplished.  Thus,  the  ad- 
ministration is  always  followed  by  a  temporary  diminution  (negative 
phase)  of  the  opsonic  index,  soon  followed,  if  the  dose  be  not  too 
large,  by  a  marked  increase  (positive  phase).  It  is  supposed, 
upon  theoretic  grounds,  and  proved  by  practical  experience,  that 
the  increase  of  phagocytic  activity  brings  about  improvement. 
The  care  of  the  operator  should  be  to  avoid  giving  so  large  a  dose  of 
the  vaccine  that  the  negative  phase  will  be  so  long  continued  that 
harm  instead  of  good  may  be  achieved. 

Although  Wright  is  said  to  cling  to  the  study  of  the  opsonic 

*  "Lancet,"  1902,  i,  p.  73. 
t  "Medicine,"  April,  1906. 


278  The  Phagocytic  Power  of  the  Blood 

index  as  a  guide  to  bacterio-vaccination  and  the  resulting  degree 
of  immunity,  the  greater  number  of  workers  have  abandoned  it 
upon  grounds  which  the  writer  long  ago  expressed — "that  the 
estimation  of  the  value  of  bacterio-vaccination  by  means  of  the 
opsonic  index  was  a  very  complicated  way  of  finding  out  very 
little." 


CHAPTER  XX 

THE  WASSERMANN  REACTION  FOR  THE  DIAGNOSIS  OF 

SYPHILIS 

THIS  now  popular  and  fairly  reliable  method  for.  assisting  in 
the  diagnosis  of  atypical  syphilitic  infections  was  devised  by  Wasser- 
mann,  Neisser,  and  Bruck.*  It  is  a  method  of  making  the  diagnosis 
of  syphilis  by  demonstrating  in  the  blood  (cerebrospinal  fluid,  milk, 
or  urine)  of  the  patient  a  complement-fixing  substance  (antibody?) 
not  present  in  normal  blood. 

The  test  is  twofold:  (i)  A  combination  of  syphilitic  antigen, 
complement,  and  suspected  serum.  (2)  A  subsequent  addition  to 
the  mixture  of  blood-corpuscles  and  hemolytic  amboceptor.  If  the 
suspected  serum  contain  the  syphilitic  antibody  the  antigen  and 
complement  unite  with  it,  and  the  complement  being  thus  "fixed," 
no  hemolysis  can  take  place  upon  the  subsequent  addition  of  the 
blood-corpuscles  and  hemolytic  serum.  If,  on  the  other  hand,  the 
suspected  serum  contain  no  antibody,  the  complement  cannot  be 
fixed,  and  is,  therefore,  free  to  act  upon  the  subsequently  added 
blood-corpuscles  in  the  presence  of  the  hemolytic  serum,  and  hemo- 
lysis results. 

It  is  thus  seen  that  the  first  test  is  made  for  the  purpose  of  fixing 
the  complement,  and  the  second  for  the  purpose  of  finding  out 
whether  it  has  been  fixed  or  not. 

It  is  quite  clear  that  such  a  test  is  very  delicate,  and  can  only 
be  successful  when  executed  with  great  precision  and  with  reagents 
or  factors  titrated,  so  that  their  exact  value  may  be  known. 

CONSIDERATION  OF  THE  REAGENTS  EMPLOYED 

I.  For  the  first,  or  fixation,  test  it  is  necessary  to  bring  together — 

Syphilitic  antigen. 

Serum  to  be  tested. 

Complement. 

(i)  The  Syphilitic  Antigen. — It  was  supposed  by  Wassermann, 
Neisser,  and  Bruck,  who  first  devised  the  test,  that  the  syphilitic 
antigen  must  contain  the  essential  micro-organisms  of  syphilis.  No 
method  for  the  cultivation  of  Treponema  pallidum  having  at  that 
time  been  devised,  cultures  of  the  specific  micro-organism  could  not 
be  employed.  Histologists  had,  however,  shown  that  greater  num- 
bers of  the  organisms  were  to  be  found  in  the  livers  of  the  congen- 
itally  syphilitic  stillborn  infants  than  anywhere  else.  With  the 

*  "Deutsch.  Med.  Wochenschr.,"  1906,  No.  19. 
279 


280         Wassermann  Reaction  for  Diagnosis  of  Syphilis 

purpose,  therefore,  of  securing  the  greatest  possible  number  of  micro- 
organisms for  the  antigenic  function,  such  livers  were  used.  The 
tissue,  having  been  cut  into  small  fragments,  was  spread  out  in 
Petri  or  other  appropriate  dishes  and  dried,  and  the  fragments 
rubbed  to  a  fine  powder  with  a  mortar  and  pestle.  Such  a  powder 
can  be  kept  indefinitely  in  an  exsiccator  over  calcium  chlorid  if 
placed  where  it  is  cool  and  dark.  When  the  powder  is  to  be  used, 
0.5  gm.  is  extracted  either  at  room  temperature  or  in  the  ice-box 
with  25  cc.  of  95  per  cent,  alcohol  for  twenty-four  hours,  filtered 
through  paper,  and  the  filtrate  used  in  quantities  later  to  be 
mentioned. 

Instead  of  drying  the  liver  tissue,  pulverizing,  and  then  extracting 
it,  many  investigators  now  prefer  to  cut  it  up,  rub  it  into  a  uniform 
paste  with  a  mortar  and  pestle,  and  add  5  volumes  of  95  per  cent. 
or  absolute  alcohol,  with  which  the  paste  is  thoroughly  macerated 
and  shaken  many  times  or  in  a  shaking  machine.  The  alcohol  may 
then  be  filtered  off,  or  may  be  permitted  to  remain  upon  the  sedi- 
mented  liver  tissue  remnants,  and  the  clear  supernatant  fluid 
pipeted  off  and  diluted,  at  the  time  of  employment,  with  the  isotonic 
sodium  chlorid  solution.  When  this  alcoholic  extract  is  added  to 
the  salt  solution  a  turbidity  occurs,  but  this  must  not  be  filtered  out, 
as  it  consists  of  the  lipoids  or  other  substances  in  the  extract  that 
are  essential  to  the  test,  and  the  quantity  of  the  cloudy  fluid  in  the 
final  mixtures  is  so  small  as  not  in  any  way  to  interfere  with  the 
results.  The  small  amount  of  alcohol  in  the  diluted  extract  is 
negligible  and  has  no  influence  upon  the  reagents  used  for  the  test. 

The  mention  of  the  lipoids  now  brings  us  to  the  point  where  it 
seems  advisable  to  state  that  one  of  the  most  interesting  facts  about 
the  Wassermann  reaction  is  that  its  theoretic  basis  was  founded  upon 
the  erroneous  assumption  that  the  essential  antigenic  substance 
consisted  of  the  whole  or  fragmented  treponemata  in  the  liver  ex- 
tract. The  method  scarcely  began  to  meet  with  practical  applica- 
tion, however,  before  it  was  discovered  that  the  active  antigenic 
substance  was  soluble  in  alcohol,  was  present  in  other  than  syphilitic 
livers,  and  could  be  extracted  not  only  from  human  tissues,  but  also 
from  dogs'  livers  and  from  guinea-pigs'  hearts.  Forges  and  Meier, 
indeed,  found  that  lecithin  could  play  the  role  of  syphilitic  antigen, 
and  Leviditi  and  Yamanouchi  place  sodium  glycocholate,  sodium 
taurocholate,  protogon,  and  cholin  among  those  bodies  capable  of 
acting  as  syphilitic  antigens,  and  Noguchi  goes  so  far  from  the  orig- 
inal that  he  regularly  employs  an  extract  of  the  normal  guinea-pig's 
heart  as  the  antigen  to  be  employed  in  his  modification  of  the  test. 

These  discoveries  now  make  it  clear  that  the  complement  fixation 
that  takes  place  in  syphilis  is  not  identical  with  that  of  the  Bordet- 
Gengou  reaction,  in  which  it  had  its  beginning.  Happily,  however, 
the  error  does  not  destroy  the  usefulness  of  the  method  for  diagnosis. 

The  probable  nature  of  the  reaction  will  be  described  below.     For 


The  Serum  to  be  Tested 


281 


the  present  we  must  be  content  to  follow  the  beaten  path,  and  for 
this  purpose  will  use  the  congenitally  syphilitic  liver  extract  as  the 
antigen,  preparing  it  as  described  above. 

(2)  The  Serum  to  be  Tested. — Wassermann,  Neisser,  and  Bruck 


Fig.  103.— The  Kei- 
del  tube  for  collecting 
blood.  (Manufac- 
tured by  the  Steele 
Glass  Co.,  of  Phila- 
delphia.) 


Fig.  104.— Parts  of  the  Keidel  tube.  E  is  the 
vacuum  bulb  which  is  attached  to  the  needle  by  a 
piece  of  rubber  tubing  (Z>);  the  glass  tube  (B) 
covers  the  needle  and  the  whole  is  sterilized. 
(Kolmer.) 


at  first  employed  the  cerebrospinal  fluid,  but  now  the  blood-serum 
of  the  suspected  patient  is  almost  universally  used.  As  is  usual 
with  antibodies,  the  substances  engaging  in  the  complement-fixation 
test  are  widely  distributed  throughout  the  body,  and  reach  the 


282         Wassermann  Reaction  for  Diagnosis  of  Syphilis 

cerebrospinal  fluid,  the  milk,  the  urine,  and  the  other  body  fluids 
through  the  blood,  in  which  it  exists  in  greatest  concentration.  The 
blood  is,  moreover,  readily  obtainable  for  study,  which  is  another 
reason  it  is  at  present  used  for  making  the  test  under  all  ordinary 
circumstances.  Noguchi,  who  works  with  very  small  quantities  of 
the  reagents,  secures  the  blood  by  obstructing  the  venous  circulation 
of  the  thumb  or  of  a  finger  by  means  of  a  rubber  band  (see  directions 
for  obtaining  the  blood  for  making  the  opsonic  index)  but  the  greater 
number  prefer  to  obtain  it  by  introducing  a  large  hypodermic  needle 
into  one  of  the  veins  near  the  bend  of  the  elbow.  The  arm  above 
the  elbow  is  compressed  by  a  fillet,  as  though  for  the  purpose  of 
performing  phlebotomy,  and  a  conspicuous  vein  selected  for  the 
purpose.  The  skin  is  first  carefully  washed,  then  treated  with 
tincture  of  iodin.  If  the  patient  is  nervous,  a  momentary  spraying 
with  chlorid  of  ethyl  will  make  the  operation  entirely  painless. 
Some  prefer  to  use  the  iodin  without  the  preliminary  washing,  be- 
lieving that  soap  makes  it  difficult  for  the  iodin  to  effect  satisfactory 
disinfection  of  the  skin.  The  sterilized  needle  is  thrust  into  the 
vein,  care  being  taken  that  the  vein  is  not  too  compressed  and  the 
point  of  the  needle  thrust  entirely  through  instead  of  into  it.  From 
15  to  25  cc.  of  blood  may  be  withdrawn  in  a  Keidel  tube,  or  into  a  large 
syringe  or  may  be  allowed  to  flow  into  a  sterile  test-tube.  The  blood, 
however  secured,  is  permitted  to  coagulate  and  the  clear  serum  re- 
moved by  a  pipette,  or  the  clotted  blood  is  placed  in  a  centrifuge 
tube  and  whirled,  so  that  clear  serum  is  secured  in  a  few  minutes. 

As  normal  human  blood-serum,  when  fresh,  contains  a  certain 
amount  of  complement  which  would  interfere  with  the  success  of 
the  experiment,  the  serum  is  next  placed  in  a  test-tube  and  kept 
in  a  water-bath  between  55°  to  58°C.  for  a  half -hour.  This  degree 
of  heat  destroys  the  complement  and  leaves  the  complement-fixing 
substance  uninjured.  The  serum  is  now  ready  for  use. 

(3)  The  Complement. — The  complement  generally  employed  is 
contained  in  the  blood  of  a  healthy  adult  guinea-pig.  To  obtain 
it  a  piece  of  cotton  moistened  with  ether  or  chloroform  is  held  to 
the  guinea-pig's  nose  until  it  becomes  unconscious,  when  the  head 
is  forcibly  extended  and  a  longitudinal  incision  made  through  the 
skin  of  the  neck.  The  skin  is  then  drawn  back  between  the  finger, 
on  the  one  side,  and  the  thumb,  on  the  other  side,  of  the  operator's 
left  hand,  while,  with  a  sharp  knife  held  in  the  right  hand,  he  cuts 
through  all  the  tissues  of  the  neck  down  to  the  spinal  column  and 
thus  opens  both  carotid  arteries.  The  spurting  blood  is  caught  in 
a  sterile  Petri  dish  and  the  animal  permitted  to  bleed  to  death. 
The  blood  soon  coagulates  when  undisturbed,  and  in  a  short  time 
clear  serum  exudes  from  the  clot.  As,  however,  the  complement 
seems  to  be  at  least  in  part  derived  from  the  corpuscles,  the  serum 
should  not  be  removed  as  soon  as  it  forms,  but  permitted  to  remain 
in  contact  with  the  clot  for  three  hours.  If  it  is  desired  to  save 


The  Blood-corpuscles  283 

time,  the  clot,  as  soon  as  formed,  may  be  cut  into  strips  and  placed 
in  the  tubes  of  a  centrifuge  and  whirled  for  a  half -hour.  This  se- 
cures a  greater  quantity  of  the  serum  and  at  the  same  time  gives  it 
its  full  value,  probably  by  injuring  the  leukocytes. 

Such  serum  containing  the  complement  is  useful  for  twenty-four 
hours.  Longer  it  should  not  be  kept  or  used,  as  it  begins  to  deterio- 
rate almost  at  once,  and  the  deterioration  increases  in  rapidity  in 
proportion  to  the  length  of  time  it  is  kept.  The  quantity  of  the  com- 
plement in  the  serum  of  the  guinea-pig  is  fairly  constant,  when  the 
animal  is  regularly  fed,  and  furnishes  a  fairly  uniform  reagent  that 
requires  no  titration. 

II.  For  the  second,  or  hemolytic,  test  two  additional  reagents 
are  required: 

Blood-corpuscles  to  be  dissolved. 

Hemolytic    amboceptors   by   which   complement   may   be 
united  to  them. 

(4)  The  Blood-corpuscles. — It  makes  no  difference  what  kind  of 
blood-corpuscles  are  employed.  Ehrlich  and  Morgenroth,  in  their 
pioneer  experiments  into  the  mechanism  of  hemolysis,  used  goat 
corpuscles.  Bordet  used  rabbit  corpuscles;  Wassermann,  Neisser, 
and  Bruck,  sheep  corpuscles;  Detre,  horse  corpuscles;  Noguchi, 
human  corpuscles. 

As  those  who  do  many  tests  require  a  considerable  quantity  of 
blood,  it  seems  wisest  to  make  use  of  some  kind  that  is  readily  ob- 
tainable in  any  quantity,  hence  most  investigators  now  follow 
Wassermann  and  his  collaborators  and  use  sheep  blood,  which  is 
easily  obtained  at  a  slaughter-house  or  from  sheep  kept  for  the 
purpose. 

The  flowing  blood  is  caught  in  some  open  receptacle,  stirred  until 
it  is  defibrinated  (it  must  not  be  permitted  to  coagulate),  and  then 
taken  to  the  laboratory. 

The  corpuscles  must  next  be  washed  with  care,  so  as  to  free  them 
from  all  traces  of  amboceptors  and  complement  belonging  to  the 
serum  in  which  they  are  contained.  For  this  purpose  a  centrifuge 
is  indispensable.  The  tubes  of  the  apparatus  are  filled  with  the 
defibrinated  blood  and  then  whirled  for  fifteen  minutes  until  the 
corpuscles  form  a  compact  mass  below  a  fairly  clear  serum.  The 
serum  is  then  cautiously  removed  and  replaced  by  0.85  per  cent, 
sodium  chlorid  solution,  the  top  of  each  tube  closed  by  the  thumb, 
and  vigorously  shaken  so  as  to  distribute  the  corpuscles  throughout 
the  newly  added  fluid.  The  tubes  are  next  returned  to  the  centrifuge 
and  again  whirled  until  the  corpuscles  are  sedimented,  when  the 
fluid  resulting  from  this  first  washing  is  removed  and  replaced  by 
fresh  salt  solution,  in  which  the  corpuscles  are  again  thoroughly 
shaken  up.  They  are  now  again  whirled  until  again  sedimented, 
when  the  second  washing  is  removed,  leaving  the  corpuscular  mass 
undisturbed.  Some  prefer  to  give  the  corpuscles  a  third  washing, 


284        Wassermann  Reaction  for  Diagnosis  of  Syphilis 

but  it  does  not  seem  to  be  necessary.  Of  the  remaining  corpuscular 
mass,  5  cc.  are  added  to  95  cc.  of  salt  solution  to  make  a  5  per  cent, 
volume  suspension,  in  which  form  they  are  ready  for  use.  As  the 
corpuscles  of  healthy  sheep  thus  treated  form  a  practically  invariable 
unit,  no  titration  or  other  preliminary  is  needed  before  they  are  used. 
They  must,  however,  be  used  within  seventy-two  hours  to  secure 
satisfactory  results,  as  they  tend  to  soften  when  kept  and  so  to  lose 
their  standard  value.  If  kept  longer  than,  twenty-four  hours  they 
should  be  washed  before  using. 

(5)  The  Hemolytic  Amboceptor. — As  the  validity  of  the  test  de- 
pends upon  the  ability  or  inability  of  the  complement  to  dissolve 
the  corpuscles,  and  as  this  can  only  be  achieved  when  appropriate 
amboceptors  are  added,  the  hemolytic  amboceptors  must  correspond 
to  the  kind  of  blood-corpuscles  employed  in  the  experiment.  As  has 
been  shown,  the  greater  number  of  investigators  now  employ  sheep 
corpuscles,  hence  must  use  such  corpuscles  as  the  antigen  through 
whose  stimulation  the  amboceptors  or  antibodies  are  excited. 

The  usual  method  of  obtaining  the  amboceptor  is  in  the  blood- 
serum  of  an  experimentally  manipulated  rabbit.  A  large  healthy 
rabbit  is  employed  for  the  purpose,  and  is  given  a  series  of  intra- 
peritoneal  injections  of  the  5  per  cent,  suspension  of  washed  and 
sedimented  sheep  corpuscles  prepared  as  above  described.  These 
injections  are  usually  given  about  five  days  apart,  and  the  dosage 
is  usually  5,  10,  15,  20  and  25  cc.  respectively. 

A  serum  of  higher  amboceptor  content  may  be  prepared  by  using 
a  greater  number  of  corpuscles,  and  for  this  purpose  the  solid  cor- 
puscular mass  thrown  down  by  centrifugalization  after  the  second 
washing  is  employed.  Of  this,  2,  4,  8,  and  12  cc.,  diluted  with  just 
enough  salt  solution  to  make  it  pass  readily  through  the  hypodermic 
needle,  may  be  regarded  as  appropriate  doses,  the  intervals  being 
the  same,  viz.,  five  days.  The  amboceptor  content  of  the  rabbit 
serum  seems  to  be  greatest  about  the  ninth  or  tenth  day  after  the 
last  injection.  Much  care  must  be  taken  to  see  that  the  injected 
fluid  is  sterile  and  the  operations  performed  under  aseptic  precau- 
tions, as  the  rabbits  are  easily  infected  and  not  infrequently  die. 
They  also  seem  prone  to  die  after  the  last  injection,  so  that  it  is  best 
to  have  more  than  one  rabbit  under  treatment  at  a  time. 

When  the  appropriate  time  has  arrived,  the  rabbit  is  bled  from 
the  carotid  artery,  according  to  the  directions  given  in  the  chapter 
upon  Experiments  upon  Animals. 

The  blood  thus  obtained  is  permitted  to  coagulate,  and  the  serum, 
which  should  be  clear,  removed  with  a  pipette.  More  serum  may  be 
obtained  from  the  clot  by  cutting  it  into  strips,  placing  these  in  a 
centrifuge  tube,  and  whirling  them  for  fifteen  minutes. 

Having  thus  described  the  preparation  of  the  reagents  to  be  em- 
ployed in  making  the  Wassermann  test,  the  next  step,  that  of  titrat- 
ing them,  becomes  essential.  One  of  the  first  questions  that  pre- 


The  Hemolytic  Amboceptor  285 

sents  itself  is  how  successful  titration  of  reagents  that  may  all  be 
more  or  less  variable  can  be  effected.  To  achieve  this  it  is  necessary 
to  begin  with  those  that  can  be  assumed  to  be  least  variable  and 
work  up  to  those  that  are  most  so. 

(1)  The  Sheep  Corpuscles. — As  these  come  from  a  healthy  animal, 
are  always  treated  in  precisely  the  same  manner  and  used  under 
standard  conditions  of  freshness,  they  can  be  looked  upon  as  an  in- 
variable factor,    i  cc.  of  the  5  per  cent,  suspension  forms  a  good 
working  quantity  and  constitutes  the  unit. 

(2)  The  Normal  Guinea-pig  Serum  Containing  the  Complement. — 
As  this  also  comes  from  a  normal  animal,  is  always  treated  in  pre- 
cisely the  same  manner,  and  is  also  used  under  standard  conditions 
of  freshness,  etc.,  it  may  also  be  looked  upon  as  a  factor  subject 
to  very  slight  variation.     Of  this  serum,  o.i  cc.  (i  cc.  of  a  i  :io 
dilution,  made  with  physiological  salt  solution)  forms  the  unit,  or 
working  quantity. 

These  two  reagents,  therefore,  may  be  regarded  as  the  standards 
of  measurement  through  which  the  titer  of  a  third  is  made  possible. 

(3)  The  hemolytic  serum  from  the  rabbit  treated  with  the  sheep 
corpuscles. 

This  is  subject  to  very  great  variation,  according  to  the  treat- 
ment of  the  rabbit,  and  apparently,  also,  according  to  the  ability 
of  the  individual  rabbit  to  respond  to  the  treatment  by  the  forma- 
tion of  hemolytic  amboceptors.  It  is,  therefore,  imperative  to  make 
a  careful  titration  of  it. 

To  do  this  we  proceed  as  follows,  the  quantities  recommended 
being  such  as  experience  has  proved  most  satisfactory: 

Into  each  of  a  series  of  common  test-tubes  or  culture- tubes  i  cc. 
of  the  5  per  cent,  suspension  of  sheep  corpuscles  and  i  cc.  of  the 
i  :  10  dilution  of  the  normal  guinea-pig  serum  (complement)  are 
measured  with  graduated  pipettes,  and  then  to  each  of  these  tubes 
the  rabbit  serum  (amboceptor),  diluted  with  physiological  salt  solu- 
tion so  as  to  make  the  correct  measurement  of  the  minute  quantities 
necessarily  employed  a  matter  of  ease  and  convenience,  is  added  in 
diminishing  quantities  for  the  purpose  of  determining  the  least 
quantity  that  will  bring  about  complete  hemolysis  in  two  hours  at 
the  temperature  of  37°C.  The  occurrence  of  the  hemolysis  is  shown 
by  a  very  striking  change  in  the  appearance  of  the  fluids.  The 
mixture  is  at  first  opaque  and  pale  red,  but  after  hemolysis,  or  solu- 
tion of  the  red  corpuscles,  becomes  a  beautiful  transparent  Burgundy 
wine  red. 

The  actual  " set-up"  or  working  scheme  for  determining  the  unit 
or  least  hemolyzing  addition  of  the  amboceptor  serum  may  be 
represented  as  follows,  the  tubes  being  placed  in  a  thermostat  and 
observed  every  fifteen  minutes: 


286         Wassermann  Reaction  for  Diagnosis  of  Syphilis 


Five  per  cent,  suspen-     Normal  guinea-pig  Hemolytic  rabbit   Result  (final  readings 

sion  of  corpuscles.  serum.  serum.  after  two  hours). 

icc.  o.     cc.  o.oi        cc.         Complete  hemolysis. 

i  o  0.005 

i  o.  0.002 

i  o.  o.ooi 

i  o .  o . 0005 

i  o.  0.0003  Partial 

i  o.i  0.0002  No 

i  o.i 


o . 0003 

O.OOO2 
0.0001 


After  the  reagents  are  added,  enough  0.85  per  cent,  salt  solution 
is  added  to  each  tube  to  bring  the  total  bulk  of  the  mixture  up  to 
5cc. 

From  the  results  shown  in  the  tubes  it  is  evident  that  the  hemolyz- 
ing  quantity  of  the  rabbit  serum  lies  between  0.0005  and  0.0003 
cc.,  and  is  probably  0.0004  cc.  To  be  as  accurate  as  possible,  a 
second  series  of  experiments  should  be  made  with  0.0005,  0.00045, 
and  0.0004  cc.,  so  that  the  proportion  of  amboceptor  serum  neces- 
sary to  effect  hemolysis  be  known  within  small  limits.  This  least 
quantity,  that  will  certainly  cause  hemolysis  in  two  hours  at  37° 
C.,  is  known  as  the  unit.  The  combination  of  the  unit  of  corpuscular 
suspension  (i  cc.),  the  unit  of  complement  (o.i  cc.),  and  the  unit 
of  hemolytic  amboceptor  is  known  as  the  hemolytic  system. 

As  soon  as  this  unit  is  known  accurately,  we  are  in  position  to 
reverse  the  conditions  of  the  test.  Thus,  if  we  should  desire  to  know 
how  much  variation  there  may  be  in  the  complements  from  different 
animals  under  different  conditions  of  age,  feeding,  health,  etc.,  we 
can  now  do  so  by  determining  whether,  when  i  cc.  of  the  corpuscles, 
i  unit  of  amboceptor  and  varying  quantities  of  complementary 
serums  are  combined,  any  variation  in  the  final  results  will  obtain. 

Or,  if  we  desire  to  know  to  what  extent  the  sheep  corpuscles  may 
change  through  prolonged  keeping  or  other  manipulation,  it  can 
be  done  by  maintaining  the  unit  of  amboceptor  and  the  unit  of 
complement  and  adding  larger  or  smaller  quantities  of  the  corpuscles. 

The  conditions  under  which  the  unit  of  amboceptor  is  titrated 
constitute  the  standard  conditions  of  the  Wassermann  reaction. 
In  it  are  always  employed  i  unit  of  sheep  corpuscle  suspension,  i 
unit  of  complement,  and  i  unit  of  amboceptor.  Here,  however,  a 
slight  difference  of  opinion  is  reached,  it  being  argued  by  many  experi- 
menters that  such  exact  proportions  may  make  the  test  uncertain, 
because,  should  there  be  the  slightest  tendency  on  the  part  of  the 
remaining  reagents  to  inhibit  hemolysis  by  means  other  than  comple- 
ment fixation,  it  would  result  in  positive  readings  where  the  final 
result  should  be  negative.  To  overcome  this  possibility,  they  dif- 
ferentiate between  the  amboceptor  unit  and  the  amboceptor  dose, 
the  latter  being  commonly  twice  and  sometimes  four  times  the 
unit. 

Now,  though  the  amboceptor  unit  is  determined  by  the  method 
given,  it  by  no  means  follows  that  those  proportions  are  the  only 


The  Hemolytic  Amboceptor  287 

ones  that  will  lead  to  hemolysis.  By  increasing  the  amboceptor 
we  can  diminish  the  complement  with  the  same  end-result,  a  matter 
that  has  been  graphically  shown  by  Noguchi,*  who  says  "that 
hemolysis  is  merely  the  relative  expression  of  the  combined  action 
of  amboceptor  and  complement,  and  is  not  the  absolute  indication 
of  the  amount  of  the  hemolytic  components  present  in  the  fluid. 
The  same  amount  of  hemolysis  can  be  produced  by  i  unit  of  com- 
plement and  by  i  unit  of  amboceptor  as  by  20  units  of  amboceptor 
and  o.i  unit  of  complement  or  any  other  appropriate  combination 
of  these  two  components." 

As  in  the  performance  of  the  test  we  work  always  with  i  unit  of 
complement,  we  do  not  want  to  unduly  disturb  its  proper  propor- 
tional action  by  any  excessive  addition  of  amboceptor,  but  simply  to 
increase  the  latter  sufficiently  to  provide  for  the  accidental  presence, 
in  the  serum  to  be  tested,  of  substances  affecting  hemolysis.  Fortu- 
nately, means  are  provided  for  controlling  this  action,  as  will  be 
shown  below. 

The  amboceptor  serum  keeps  indefinitely.  When  it  is  to  be  kept 
and  used  from  time  to  time,  many  experimenters  prefer  to  seal 
it  in  a  number  of  small  tubes,  one  of  which  is  opened  when  the 
serum  is  needed,  the  remainder  being  kept  in  an  ice-box.  Others 
prefer  a  stoppered  bottle  that  can  be  opened  and  a  measured  quan- 
tity removed  as  needed.  The  most  convenient  way  of  treating  it 
seems  to  be  Noguchi's  method  of  drying  it  upon  filter-paper. 

For  this  purpose  a  good  quality  of  filter-paper  is  cut  into  strips 
10  to  20  cm.  in  length  and  6  to  8  cm.  in  breadth,  and  saturated  with 
the  serum,  which  is  permitted  to  dry.  It  is  well  to  make  a  pre- 
liminary titration  of  the  serum,  for  if  it  be  very  active  it  may  have 
to  be  diluted  in  order  that  the  piece  of  dry  paper  containing  the  dose 
be  of  a  size  convenient  to  handle;  i  drop  of  serum  usually  covers 
about  %  scl-  cm*>  which  is  about  as  small  a  piece  as  can  be  measured, 
cut,  and  used  with  satisfaction  if  sufficient  allowances  are  to  be 
made  for  variations  in  distribution  and  other  conditions  that  may 
modify  the  accuracy  of  the  method.  If  the  unit-strength  of  a  serum 
be,  say,  0.00005  and  the  dose  o.oooi,  water  should  be  added  to  the 
extent  of  about  9  volumes  and  the  mixture  gently  agitated,  so  that 
diffusion  may  occur  without  frothing.  The  diluted  serum  is  poured 
into  a  large  flat  dish,  and  the  strips  of  paper  passed  lengthwise  and 
slowly  to  and  fro  until  not  only  wet,  but  thoroughly  saturated. 
Each  strip,  when  the  dipping  is  finished,  is  held  first  by  one  end, 
then  by  the  other,  to  drain  off  the  free  drops,  and  then  laid  flat 
upon  a  clean  glass  plate  and  permitted  to  dry.  The  use  of  an  electric 
fan  is  recommended  to  hasten  drying.  Paper  so  prepared  contains 
everywhere  about  the  same  quantity  of  serum. 

The  real  titration  of  the  serum  now  begins.  With  a  ruler,  one  piece  of  paper 
is  divided  into  squares  of,  say,  ^  cm.,  and  a  series  of  tubes  prepared  with  cor- 

*  "Serum  Diagnosis  and  Syphilis,"  1910,  p.  13  et  seq. 


288        Wassermann  Reaction  for  Diagnosis  of  Syphilis 

puscle  suspension  and  complement  and  the  paper  added  i  square,  2  squares, 
2^  squares,  and  so  on  until  the  unit  is  determined.  When  that  is  achieved, 
the  exact  size  of  the  paper  containing  the  unit  being  known,  one  sheet  of  the 
paper  can  be  ruled  into  squares  of  that  size  or  into  squares  of  twice  that  size — • 
since  the  "dose"  is  two  units — at  the  option  of  the  investigator. 

The  sheets  of  paper  are  kept  in  a  clean  envelope,  the  quantity 
for  each  test  being  cut  off  as  needed.  The  dry  serum  changes  so 
little  that  the  dose  once  determined,  the  size  of  the  square  of  paper 
needed  for  the  test  remains  about  the  same. 

The  method  has  the  advantage  that  the  amboceptor  serum 
cannot  be  spoiled  or  spilled.  It  has  the  disadvantage  of  being 
slightly  less  accurate,  though  it  must  be  admitted  that  the  chances 
of  error  in  measuring  and  diluting  the  fluid  serum  are  probably  as 
great  as  those  arising  from  inequalities  in  the  distribution  of  the 
serum  throughout  the  paper. 

(4)  The  Antigen. — It  has  already  been  shown  that  complement 
is  labile,  and  it  may  have  occurred  to  the  reader  that  its  activity 
is  similar  to  that  of  ferments.  It  is  now  necessary  to  point  out  the 
many  conditions  (some  of  which  may  arise  in  the  performance  of 
a  test  so  delicate  as  the  Wassermann  reaction)  by  which  the  comple- 
mentary action  may  be  affected  or  set  aside.  Thus,  temperature 
affects  it,  and  temperatures  of  o°C.  suspend  it.  It  is  on  this  ac- 
count that  the  test  is  always  made  at  37°C.  Like  most  of  the 
ferments  of  the  living  organism,  salts  affect  it,  and  in  salt-free  media 
its  action  ceases,  to  return  when  a  small  quantity  of  an  alkaline  salt 
is  added.  Not  only  inorganic  salts,  but  salts  of  the  fatty  acids  and 
the  bile-salts  may  inhibit  it.  Certain  lipoids,  such  as  lecithin, 
cholesterin,  protogon  and  tristearin,  and  neutral  fats  inhibit  the 
complementary  action.  Some  of  these  substances  are  always 
present  in  the  serum  containing  the  complement  itself  or  in  the  other 
serums  to  be  tested  by  its  use,  and,  as  Wassermann  and  Citron  have 
pointed  out,  we  really  know  nothing  about  complementary  action. 
Aleuronat,  inulin,  peptone,  albumose,  tuberculin,  natural  and 
artificial  aggressins,  gelatin,  casein,  sitosterin,  coagulated  serum- 
albumin,  and  albuminous  precipitates  all  act  as  inhibitives  to 
complementary  action. 

Now,  in  all  combinations  of  several  serums  and  antigens  it  is 
always  possible  that  some  of  these  complement-binding  or  comple- 
ment-inhibiting substances  may  be  present,  hence  the  first  thing  that 
has  to  be  done  in  the  way  of  titrating  the  antigen — which  is  a  tissue 
extract,  rich  in  lipoids  which  inhibit  complementary  action — is  to 
determine  how  much  of  it  can  be  added  to  the  "hemolytic  system" 
without  disturbing  hemolysis. 

As,  however,  the  antigen  is  not  used  by  itself,  but  always  in  com- 
bination with  a  serum  to  be  tested,  we  must  always  combine  it  with 
serum  when  making  the  titration,  so  that  the  requirements  of  the 
test  may  be  conformed  with.  In  order  that  the  essential  difference 
between  the  normal  serum  and  the  syphilitic  serum  can  be  reduced 


The  Hemolytic  Amboceptor 


289 


to  precise  calculation  it  is  imperative  that,  in  all  the  tests,  the  same 
quantity  of  added  serum  be  employed.  Experience  has  shown  this 
quantity  to  be  0.2  cc.,  and  this  we  regard  as  the  unit  of  serum  to  be 
tested. 

To  titrate  the  antigen  we  require  (i)  a  normal  human  serum  and 
(2)  a  known  syphilitic  serum,  obtained  from  blood  drawn  from  the 
arm  veins  of  cases  known  to  be  well  and  cases  known  to  be  syphilitic 
respectively.  These  serums  should  be  kept  on  hand  in  the  labora- 
tory in  considerable  quantity,  as  they  are  constantly  needed  for 
making  the  controls  that  must  accompany  each  test,  as  well  as  for 
making  the  preliminary  titration  of  the  antigen. 


Tubes 
i.       i  unit  of 


TABLE  I.  —  Series  with  the  Normal  Serum 


i  unit  of      +  antigen  o.oi 
complement       normal  serum 
2.  "  +  "  +        ''0.03 


Complete 
hemolysis. 


3-                         +                           ~r 

O.O5          j-j  rt  w^^ 

— 

o;  bi  5  >j  D 

4-                           +                             + 

°  .07            "5  a;   g  O  60 

=             *  ' 

5.         '"       +         "         + 

"         /->    ^Q          nn  "^  "o  <"  ^ 
O  .  Oo            o  J3       G  O 

= 

6.                          +                            + 

"    0.09     ^o^£j5 

= 

7-                          +             "             + 

c<       o.oi       -oJllS 

= 

8.                         +                           + 

"       0.12        Gg-u-Sj 

= 

9-                          +                            + 

"    o.is    2i||| 

1  1 

10.                                     +                                        + 

"       0.18       |^l|^ 

_              t  ( 

ii.             "           +                            + 

"     0.2      ""fe'g'ajr! 

=     No 
hemolysis. 

TABLE  II.—  Series 

wi//f  ^Ae  Syphilitic  Serum 

Tubes 

i.      i  unit  of     +       i  unit  of       + 
complement      syphilitic  serum 

antigen  o.oi         ^  §  I'^'w 

=     Complete 
hemolysis. 

2.                  "             .+                  "                 + 

"  °-°3    lisll 

=              " 

3-             "           +                            + 

11  0.05    lifli 

=     Sugges- 

« rt  S  «  M 

tion  of 

"S  «  g-5.S 

hemolysis. 

4.             "           +                            -f 

"      0.07        -25^°S 

=     Slight 

Td^cj  °'g  ^ 

hemolysis. 

5'            "          +            "            + 

"          0.08              2  rt  §  S.S 

=      Partial 

M   «^2    & 

a;  O      "£^0 

hemolysis. 

6.                        +                          + 

"       0.09          «  a  "  «§ 

=     No 

•oJI  S« 

hemolysis. 

7.            "          +            "            + 

°'J     'illli 

= 

8.                        +                          + 

"    0.12      (fs!.s* 

=             " 

9.                        -f                          + 

"     0.15       "ggll^ 

= 

10.                                   +                                      + 

"       0.18         HQ^U^ 

—             '  ' 

290        Wassermann  Reaction  for  Diagnosis  of  Syphilis 

The  " set-up"  for  the  titration  of  antigen  is  fairly  simple.  A 
series  of  tubes  is  prepared  and  divided  into  two  groups.  Into  each 
tube  in  each  group  is  placed  i  unit  of  complement.  Each  tube  of 
one  group  receives  the  addition  of  0.2  cc.  of  the  normal  serum;  each 
tube  of  the  other  group,  0.2  cc.  of  the  known  syphilitic  serum.  All 
the  tubes  now  receive  additions  of  antigen,  so  that  one  tube  of  each 
group  contains  the  same  quantity.  The  quantity  of  antigen  not 
being  known,  it  is  only  through  the  experience  of  others  that  we  can 
guess  where  to  start.  An  idea  can  be  formed  through  study  of  the 
tabulation  on  page  289. 

From  this  we  find  that  the  unit  of  antigen  is  0.09  cc.,  the 
largest  quantity  of  the  antigen  that  can  be  added  without  prevent- 
ing hemolysis  when  the  normal  serum  is  used  is  probably  0.18  cc. 
At  the  same  time  0.09  cc.  is  the  smallest  quantity  that  can  be  added, 
when  the  syphilitic  serum  is  used,  to  prevent  it.  In  this  case  the 
dose  exactly  fulfils  Kaplan's  requirement  that  "The  unit  dose  of 
antigen  must  completely  inhibit  hemolysis  .  .  .  of  a  known  luetic 
serum,  provided  double  the  dose  does  not  interfere  with  the 
complete  hemolysis  of  cells  using  a  known  normal  serum  and 
complement." 

We  have  now  accomplished  the  titration  of  all  five  of  the  factors 
involved  in  making  the  Wassermann  reaction,  but  we  have  done 
more,  we  have  really  done  the  test,  and  have  seen  positive  and 
negative  results,  for  in  titrating  the  antigen  we  have  developed  the 
reaction  by  which  we  can  confirm  the  diagnosis  of  syphilis  in  the 
case  from  whom  the  syphilitic  serum  was  obtained,  and  have  failed 
to  develop  it  with  the  known  normal  serum. 

However,  in  order  that  those  who  perform  the  test  may  be  able 
to  escape  the  numerous  errors  into  which  one  may  fall,  it  will  be 
necessary  to  point  out  the  controls  by  which  they  can  be  avoided. 

A  Wassermann  reaction  at  the  present  time  comprises  not  only 
the  test  of  the  patient's  serum,  but  simultaneously  includes  a  long 
series  of  other  tests  by  which  the  validity  of  every  part  of  the  test 
and  the  correct  titer  of  all  the  reagents  employed  can  be  simultane- 
ously ascertained.  Every  one  who  makes  the  test  should  practice 
some  such  systematic  method  as  is  suggested  by  the  following  scheme 
for  the  "  set-up."  Nine  tubes  are  employed  for  the  usual  test. 
These  are  stood  in  a  rack  in  the  same  order  for  every  test,  and  in  the 
course  of  time  it  becomes  a  matter  of  habit  to  know  the  tubes  by 
number,  and  to  recall  for  what  each  stands. 

If  many  tests  are  to  be  made  at  one  time,  it  is,  of  course,  un- 
necessary to  make  more  than  one  series  of  controls. 

Of  the  complementary  serum  we  add  i  cc.  to  9  cc.  of  0.85  per 
cent,  (physiologic)  salt  solution,  making  each  cubic  centimeter  of 
the  dilution  of  the  fluid  equal  o.i  cc.  This  quantity,  carefully 
measured-  by  the  same  volumetric  pipette,  is  dropped  into  each 
tube,  and  this  pipette  laid  aside. 


The  Hemolytic  Amboceptor 


291 


fl 


:gw 


TEST 
Tube  containing  the  serum  to  be  tested. 


Salr 
odd 


«  ti 


CONTROL 

Control  of  serum  to  be  tested  to  deter- 
mine  substances  which  without  antigen 
may  inhibit  hemolysis. 


Sell- 
add 


CONTROL 

Control  of  the  test  by  the  use  of  a 
known  syphilitic  serum. 


8^ 

« 

^•* 

*? 

<g 

$  2  ** 

* 

9  $' 

CONTROL 

^. .     ^         Control  of  the  known  positive  to  deter- 
w      3   £    mine     that     it     contains     no     recently 
"-.  "^   developed  substances  that  may  inhibit 
^   hemolysis. 


1   II 

1    II 

* 

CONTROL 

o   o^       Control  of  the  test  by  the  use  of  a 
^*  r*   known  normal  serum. 

83,3-  p; 


CONTROL 

g  Control  of  the  known  normal  serum  to 
EIS-  ^j  determine  that  no  substances  inhibiting 
rt.  p  n  hemolysis  had  developed  in  it. 

n  OJ      ^ 

r4 

««,  5 

?TS    ^ 


a  ^    e 
I?   1 


CONTROL 

^        Control  test  to  determine  changes  in 
jj.  «   the  antigen  by  which  hemolysis  might  be 
re  o    §"  prevented. 


y  g 
o  S. 
3  5' 


I! 


ll 

?  s. 


3 

§.§ 


CONTROL 

Control  of  the  hemolytic  system. 


CONTROL 

Control  for  the  purpose  of  determining 
the  presence  of  anti-sheep  amboceptors 
in  the  serum  to  be  tested. 


f  1 

r     tf- 

- 

292         Wassermann  Reaction  for  Diagnosis  of  Syphilis 

The  serum  to  be  tested  is  drawn  into  a  second  finely  graduated 
pipette,  and  0.2  cc.  added  to  tubes  i,  2,  and  9,  and  that  pipette 
laid  aside. 

The  positive  syphilitic  serum  used  to  control  the  test  is  similarly 
drawn  up  in  a  fresh  pipette  and  0.2  cc.  of  it  measured  into  tubes 
3  and  4,  and  the  pipette  laid  aside. 

The  normal  serum  used  as  a  control  is  similarly  drawn  into  still 
another  pipette  and  0.2  cc.  measured  into  tubes  5  and  6,  and  the 
pipette  laid  aside. 

The  alcoholic  extract  composing  the  antigen  is  next  added,  either 
by  diluting  it  so  that  i  cc.  contains  the  unit,  or  measuring  the  unit 
quantity  directly  into  the  tubes.  The  antigen  is  added  to  tubes 
i,  3,  5,  and  7,  and  the  pipette  laid  aside. 

Lastly,  each  tube  receives  a  correctly  measured  quantity  of  0.85 
per  cent,  sodium  chlorid  solution  to  bring  the  total  bulk  of  fluid  up 
to  exactly  3  cc. 

Each  tube  is  now  shaken  carefully,  so  as  not  to  cause  frothing  of 
the  fluid,  and  the  rack  is  stood  in  a  thermostat  kept  at  37°C. 

At  the  end  of  an  hour  the  rack  is  removed,  and  every  tube  receives 
the  addition  of  i  unit  of  the  sheep  corpuscle  suspension  and,  with 
the  exception  of  tube  9,  receives  one  dose  of  amboceptor,  either  the 
serum  measured  by  diluting  so  that  i  cc.  equals  the  dose,  or  the 
necessary  square  of  paper.  This,  in  the  former  case,  brings  the  total 
bulk  of  fluid  to  5  cc.,  in  the  latter  makes  it  necessary  to  add  i  more 
cubic  centimeter  of  salt  solution  to  each  tube.  We  aim  to  have 
exactly  5  cc.  of  fluid  in  each  tube. 

The  tubes  are  again  stood  in  the  thermostat,  where  they  are  per- 
mitted to  remain  for  an  hour,  when  the  readings  are  taken  and 
carefully  noted.  After  this  the  rack  and  all  the  tubes  are  placed 
in  the  ice-box  until  twenty-four  hours  old,  when  the  final  readings 
are  taken  and  the  conclusions  are  reached. 

As  a  rule,  the  readings  taken  after  the  second  hour  of  incubation 
and  those  taken  after  twenty-four  hours  correspond. 

A  valid  test  should  show  the  following: 

Tubes 

1.  No  hemolysis  in  syphilis.     Hemolysis  in  health. 

2.  Complete  hemolysis. 

No  hemolysis  (this  is  the  standard  of  comparison). 


Test  Controls. 


4.  Complete  hemolysis. 


9.  No  hemolysis,  as  a  rule. 

In  the  tubes  in  which  hemolysis  takes  place  the  change  is  very 
marked.  The  hemoglobin  dissolves  out  of  the  corpuscular  stroma 
and  saturates  the  fluid,  transforming  it  from  the  opaque  pale  red 
to  a  transparent  Burgundy  red.  Sometimes  the  corpuscular 


The  Hemolytic  Amboceptor 


293 


stroma  dissolves,  sometimes  it  sediments  as  a  colorless  mass  to  the 
bottom  of  the  tube. 

In  the  tubes  containing  the  positive  or  syphilitic  serum,  and  in 
which  there  is  complete  complement  fixation,  the  unaltered  cor- 
puscles sediment  to  the  bottom  of  the  tube,  leaving  a  colorless 
fluid  above. 

When  the  complement  fixation  is  complete  there  is  no  solution 
of  the  hemoglobin.  Such  a  result  has  been  described  by  Citron  as 
-f-  +  +  -f.  When  the  sedimented  corpuscles  lie  at  the  bottom  of  a 
slightly  reddened  fluid,  the  result  is  said  to  be  +  +  + ;  when  at  the 
bottom  of  a  distinctly  red  fluid,  H — h,  etc.  Confusion  will  be 
avoided  by  making  renorts  as  positive  in  all  cases  in  which  there  is 


Fig.  105. — A  typical  positive  Wassermann  reaction  with  the  recommended 
controls  as  it  appears  after  standing  twelve  hours.  Corpuscular  sedimentation 
without  hemolysis  is  seen  in  tubes  1,3,  and  9;  complete  hemolysis  in  the  others. 


a  distinct  red  corpuscular  deposit,  regardless  of  the  state  of  the 
supernatant  fluid,  and  negative  when  there  is  no  such  deposit. 

When  we  come  to  inquire  why  the  supernatant  fluid  should  be 
red,  we  reach  a  question  that  is  not  quickly  answered.  In  order 
to  be  in  a  position  to  explain  it  in  certain  cases  we  introduced  in  our 
series  tube  9,  by  which  to  discover  whether  the  serum  under  examina- 
tion contain,  as  is  sometimes  the  case  in  health  as  well  as  in  syphilis, 
sheep  corpuscle  amboceptors.  If  tube  9  shows  such  amboceptors 
to  be  in  the  serum,  it  explains  the  redness  of  the  fluid  bathing  the 
corpuscles,  and  does  not  invalidate  the  test.  If  no  such  amboceptors 
are  present  and  the  fluid  is  still  red,  it  may  indicate  that  a  little  of 


294         Wassermann  Reaction  for  Diagnosis  of  Syphilis 

the  complement  remained  unfixed  and  acted  upon  a  few  of  the 
corpuscles. 

The  Validity  of  the  Test. — The  Wassermann  reaction  is  not  a  certain 
test  for  syphilis.  It  is  an  aid  in  making  the  diagnosis,  especially 
in  cases  in  which  there  are  no  symptoms. 

Of  thousands  of  bloods  of  normal  persons  examined,  the  results 
are  almost  100  per  cent,  negative.  Basset-Smith  has  had  a  positive 
reaction  in  a  case  of  scarlet  fever  and  one  in  a  case  of  malignant 
disease  of  the  liver  with  jaundice;  Oppenheim,  one  in  a  case  of  tumor 
of  the  cerebellopontine  angle;  Marburg,  one  in  a  similar  case;  New- 
mark  reports  2  cases  of  brain  tumors  with  positive  reactions;  Cohn, 
a  positive  in  a  patient  with  a  cerebral  tumor.  The  Wassermann 
reaction  is  of  no  value  for  the  differential  diagnosis  of  syphilis  and 
framboesia  or  yaws.  All  cases  of  the  latter  give  a  positive  reaction. 
Positive  reactions  have  been  found  in  some  cases  of  nodular  leprosy, 
in  a  few  cases  of  malaria,  in  some  cases  of  pellagra,  and  in  a  good 
many  cases  of  sleeping  sickness.  These  seem  to  form  the  greater 
part  of  positive  reactions  in  non-syphilitics  thus  far  recorded. 

In  active  syphilis  Wassermann  had  90  per  cent,  of  positive  reac- 
tions in  2990  cases;  and  most  others  report  about  the  same.  Basset- 
Smith  in  458  such  cases  found  94  per  cent,  positive  reactions. 

In  latent  syphilis  Wassermann  found  50  per  cent,  positive  reactions; 
Basset-Smith,  46  per  cent. 

In  chronic,  presumably  syphilitic,  disease  of  the  nervous  system, 
general  paresis,  and  tabes  dorsalis  the  positive  reactions  vary.  In 
the  former  disease  some  have  found  as  high  as  90  per  cent,  positive; 
in  the  latter  the  usual  figures  vary  about  50  per  cent. 

It  is  thus  seen  that  the  occurrence  of  the  reaction  is  much  more 
conclusive  evidence  of  the  presence  of  syphilitic  infection  than  the 
failure  of  the  reaction  is  of  its  absence. 

Treatment  greatly  influences  the  test.  When  under  active 
treatment,  either  with  mercury  and  ipdids  or  with  salvarsan,  the 
reaction  of  the  serums  is  usually  negative. 

Nature  of  the  Reaction. — We  now  reach  the  point  of  considering 
the  nature  of  the  reaction.  It  is  certainly  not  a  variation  of  the 
Bordet-Gengou  phenomenon.  It  does  not  occur  because  of  the 
presence  in  the  blood  of  syphilitics  of  antibodies  which  combine  with 
the  antigen  and  fix  the  complement.  It  is  probably  not  comple- 
ment fixation  so  much  as  complementary  inhibition,  through  the 
presence  in  the  blood  of  syphilitics  of  certain  metabolic  products, 
whose  action  interferes  with  the  complement  in  some  entirely 
different  manner. 

NOGUCHTS  MODIFICATION  OF  THE  WASSERMANN  REACTION 

Noguchi*  has  modified  the  Wassermann  reaction,  first  by  employ- 
ing as  an  antigen  an  extract  of  the  heart  of  a  normal  guinea-pig, 
*  "Serum  Diagnosis  of  Syphilis,"  Philadelphia,  1910,  J.  B.  Lippincott  Co. 


Noguchi's  Modification  295 

and,  second,  by  making  use  of  human  instead  of  sheep  corpuscles 
for  the  hemolytic  test.  The  advantage  of  the  latter  depends  upon 
the  fact,  carefully  determined  by  Noguchi,  that  human  blood-serum 
contains  no  amboceptors  active  in  effecting  hemolysis  of  human  blood- 
corpuscles,  though  it  not  infrequently  contains  hemolytic  ambocep- 
tors for  sheep  corpuscles.  In  the  directions  for  making  the  Wasser- 
mann  test  a  control  test  for  determining  their  presence  or  absence  was 
found  expedient.  It  will  also  be  remembered  that  the  presence  of 
these  amboceptors  causes  no  invalidity  of  the  test,  provided  it  be 
recognized. 

Noguchi  also  varies  the  technic  in  such  a  manner  that  very  small 
quantities  of  the  various  reagents  are  employed — a  necessity  that 
arises  from  the  relatively  small  quantity  of  the  patient's  blood  ob- 
tainable according  to  the  method  he  employs.  The  reagents 
employed  are  as  follows: 

(1)  The  Serum  to  be  Tested. — To  obtain  this,  Noguchi  binds  the 
finger  of  the  patient  with  a  rubber  band,  makes  a  good-sized  punc- 
ture near  the  root  of  the  nail  with  a  Hagedorn  needle,  and  collects 
about  2  cc.  of  the  blood  in  a  Wright  tube  (see  directions  for  making 
the  opsonic  index).     The  blood  soon  coagulates  in  the  tube,  which  is 
then  scratched  with  a  diamond  or  file,  broken,  and  the  serum  re- 
moved with  a  capillary  pipet.     The  serum  may  or  may  not  be  in- 
activated  by  heat,   according  to  the  option  of  the  experimenter. 
The  dose  of  the  unheated  serum  is  i  drop;  of  the  inactivated  serum, 
4  drops.     The  same  doses  of  the  normal  and  syphilitic  control  serums 
are  used. 

(2)  The  Complement. — This   consists  of  fresh  guinea-pig  serum. 
Of  it  he  makes  a  40  per  cent,  dilution  in  physiologic  salt  solution  by 
adding  one  part  of  the  serum  to  ij£  parts  of  the  salt  solution;  o.i 
cc.  is  the  unit.     Two  units  constitute  the  "dose." 

(3)  The  Antigen. — The  antigen  is  made,  according  to  the  direc- 
tions given  in  the  description  of  the  Wassermann  test,  out  of  normal 
guinea-pig  heart.     The  extract  is  dried  upon  filter-paper,  as  has  been 
recommended  for  the  hemolytic  amboceptor,  and  titrated  according 
to  the  size  of  the  square  of  paper  needed,  instead  of  the  quantity  of 
fluid  to  be  added. 

(4)  The  Corpuscle  Suspension. — For  this  purpose  either  normal 
human  corpuscles  or  the  corpuscles  of  the  patient  whose  blood  is  to 
be  examined  may  be  employed.     Instead  of  a  5  per  cent,  suspension 
a  i  per  cent,  suspension  is  recommended.     If  normal  corpuscles  are 
employed,  it  is  necessary  to  wash  them  free  of  the  normal  serum  or 
plasma,  which  Noguchi  accomplishes  as  follows:  8  cc.  of  normal  salt 
solution  are  placed  in  a  large  test-tube,  and  the  blood  flowing  from 
a  puncture  (in  the  operator's  own  finger,  for  example)  permitted  to 
drop  in,  the  proportion  being  i  drop  each  4  cc.     The  fluid  is  then 
shaken  and  stood  on  ice  over  night,  when  the  corpuscle  sediment 
and  the  supernatant  fluid  containing  the  fibrin  factors  and  ferment 


296         Wassermann  Reaction  for  Diagnosis  of  Syphilis 

is  decanted  and  replaced  by  fresh  salt  solution,  and  the  suspension 
made  by   shaking.     Or,   in   a  laboratory,    the   corpuscles   can   be 


»  w  » 

K-§*'^ 

g^Cjq    g    %^ 

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Incubation  at  37°  C.  for  1  hour. 

Addition  of  antihuman  amboceptor,  2 
units  to  all  tubes. 

Incubation  at  37°  C.  for  2  hours  longer, 
then  at  room  temperature. 

washed  as  usual  with  the  aid  of  the  centrifuge.  If  the  patient's 
own  corpuscles  are  to  be  employed,  some  of  them  may  be  dis- 
tributed, through  the  serum  without  any  washing,  by  simply  shaking 


Noguchi 's  Modification  297 

it  up  a  little  with  the  clot.  It  is  not  essential  exactly  to  measure  the 
corpuscles,  as  after  a  few  trials  with  the  suspension  of  normal  cor- 
puscles the  eye  becomes  accustomed  to  the  color,  intensity,  and 
density  corresponding  to  the  requirement. 

(5)  The  Antihuman  Hemolytic  Amboceptor. — This  is  prepared  by 
injecting  rabbits,  according  to  the  method  already  described,  with 
washed  human  corpuscles  obtained  from  fresh  human  placentae 
or  from  the  heart  of  a  fresh  cadaver  come  to  autopsy.  The  serum 
of  the  rabbit,  when  obtained,  is  dried  upon  blotting-paper  and 
titrated  as  already  described. 

The  " set-up"  for  the  test,  as  given  by  Noguchi,  is  less  cumber- 
some than  that  recommended  for  the  Wassermann  test  and  includes 
six  tubes.  It  can  best  be  understood  by  reference  to  the  diagram. 

The  method  recommends  itself  through  its  simplicity  and  con- 
venience, no  sheep  corpuscles  being  used,  and  through  the  smaller 
quantity  of  blood  required,  it  seeming  to  the  patient  that  less 
damage  is  done  by  pricking  the  finger  than  by  introducing  a  syringe 
needle  into  a  vein.  It  is,  moreover,  a  very  sensitive  test,  and  gives 
very  accurate  results  as  far  as  regards  positive  cases.  Unfortunately, 
it  seems  to  have  the  demerit  of  occasionally  finding  the  reaction  in 
negative  cases,  which  is  a  serious  defect. 

Diagnosticians  are  still  divided  in  opinion,  some  preferring  the 
Wassermann  test,  some  the  Noguchi  test,  and  some  always  doing 
both,  permitting  the  one  to  control  the  other.  In  the  long  run  the 
Wassermann  test  seems  to  meet  with  most  favor,  and  in  the  hands 
of  the  majority  leads  to  most  satisfactory  results. 


PART  II 

THE  INFECTIOUS  DISEASES  AND  THE 
SPECIFIC  MICRO-ORGANISMS 


CHAPTER  I 
SUPPURATION 

SUPPURATION  was  at  one  time  looked  upon  as  a  normal  and  in- 
evitable outcome  of  the  majority  of  wounds,  and  although  bacteria 
were  early  observed  in  the  purulent  discharges,  the  insufficiency  of 
information  then  at  hand  led  to  the  belief  that  they  were  spon- 
taneously developed  there. 

From  what  has  already  been  said  about  the  evolution  of  bac- 
teriology and  the  biology  and  distribution  of  bacteria,  the  relation- 
ship existing  between  bacteria  and  suppuration,  and,  indeed,  be- 
tween bacteria  and  disease  in  general,  is  found  to  be  reversed. 
Instead  of  being  the  products  of  disease,  the  micro-organisms  are 
the  cause. 

Suppuration,  while  nearly  always  the  result  of  micro-organismal 
activity,  is  not  a  specific  infectious  process. 

Being  but  the  expression  of  tissue  irritation  arising  through  strong 
chemotactic  influences,  as  many  bacteria  may  be  associated  with  it 
as  can  bring  about  the  essential  conditions.  Bacteria  with  which 
these  qualities  are  exceptionally  marked  appear  as  the  common 
cause  of  the  process;  those  with  which  it  is  less  marked,  as  excep- 
tional causes. 

The  relative  frequency  with  which  certain  varieties  of  bacteria 
are  associated  with  suppuration  is  shown  in  the  following  table 
from  Karlinski:* 

Suppuration  in  man —                       Streptococci,  ^  45  cases. 

Staphylococci,  144 

Other  bacteria,  15 

Suppuration  in  the  lower  animals — Streptococci,  23 

Staphylococci,  45 

Other  bacteria,  15 

Suppuration  in  birds —                      Streptococci,  1 1 

Staphylococci,  40 

Other  bacteria,  20 

Andrewes  and  Gordon,  f  after  the  examination  of  large  numbers 

*  "Centralbl.  f.  Bakt.,"  etc.,  1800,  vn,  S.  113. 

t  "  Report  of  the  Local  Government  Board  of  Great  Britain,"  Supplement; 
"Report  of  the  Medical  Officers,"  1905-06,  vol.  xxxv,  p.  543- 

299 


300  Suppuration 

of  staphylococci  from  lesions  of  the  human  skin  and  mucous  mem- 
branes, came  to  the  conclusion  that  four  varieties  are  differentiate. 
Of  these,  the  Staphylococcus  pyogenes  is  the  most  common  and 
most  important.  When  typical,  it  produces  an  orange-colored  pig- 
ment; when  atypical,  it  may  be  lemon  yellow  or  white.  Staph- 
ylococcus epidermidis  albus  is  a  distinct  species.  The  differences 
between  these  cocci  are  shown  in  the  table. 

STAPHYLOCOCCUS  EPIDERMIDIS  ALBUS  (WELCH) 

General  Characteristics. — A  non-motile,  non-flagellate,  non-sporogenous, 
slowly  liquefying,  non-chromogenic,  aerobic  and  optionally  anaerobic,  doubtfully 
pathogenic  coccus,  staining  by  the  usual  methods  and  by  Gram's  method,  and 
having  its  natural  habitat  upon  the  skin. 

Under  the  name  Staphylococcus  epidermidis  albus,  Welch*  has 
described  a  micrococcus  which  seems  to  be  habitually  present  upon 
the  skin,  not  only  upon  the  surface,  but  also  deep  down  in  the  Mal- 
pighian  layer.  He  believes  it  to  be  Staphylococcus  pyogenes  albus 
in  an  attenuated  condition,  and  if  this  opinion  be  correct,  and  there 
is  seated  deeply  in  the  derm  a  coccus  which  may  at  times  cause  sup- 
puration, the  conclusions  of  Robb  and  Ghriskey,  that  sutures  of 
cat-gut  when  tightly  drawn  may  be  a  cause  of  skin-abscesses  by 
predisposing  to  the  development  of  this  organism,  are  certainly 
justifiable.  As  the  morphologic  and  cultural  characteristics  of  the 
organism  correspond  fairly  well  to  those  of  the  following  species,  no 
separate  description  of  them  seems  necessary. 

STAPHYLOCOCCUS  PYOGENES  ALBUS  (ROSENBACH)! 

General  Characteristics. — A  non-motile,  non-flagellate,  non-sporogenous, 
liquefying,  non-chromogenic,  aerobic  and  optionally  anaerobic,  mildly  patho- 
genic coccus,  staining  by  the  ordinary  methods  and  by  Gram's  method. 

Although,  as  stated,  Staphylococcus  pyogenes  albus  is  a  common 
cause  of  suppuration,  it  rarely  occurs  alone,  Passet  so  finding  it  in 
but  4  out  of  33  cases  investigated.  When  pure  cultures  of  the  coccus 
are  subcutaneously  injected  into  rabbits  and  guinea-pigs,  abscesses 
occasionally  result.  Injected  into  the  circulation,  the  staphylococci 
occasionally  cause  septicemia,  and  after  death  can  be  found  in  the 
capillaries,  especially  in  the  kidneys.  From  this  it  will  be  seen  that 
the  organism  is  feebly  and  variably  pathogenic. 

In  its  morphologic  and  vegetative  characteristics  Staphylococcus 
albus  is  almost  identical  with  the  species  next  to  be  described,  dif- 
fering from  it  only  in  the  absence  of  its  characteristic  golden 
pigment. 

*  "Amer.  Jour.  Med.  Sci.,"  1891,  p.  439. 

f  "  Wundinfektionskrankheiten  des  Menschen,"  Wiesbaden,  1884. 


Staphylococcus  Pyogenes  Albus 


301 


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302  Suppuration 

STAPHYLOCOCCUS  PYOGENES  AUREUS  (ROSENBACH*) 

General  Characteristics. — A  non-motile,  non-flagellate,  non-sporogenous' 
liquefying,  chromogenic,  pathogenic,  aerotJlc  and  optionally  anaerobic  coccus' 
staining  by  the  ordinary  methods  and  by  Gram's  method. 

Commonly  present  upon  the  skin,  though  in  smaller  numbers  than 
the  organisms  already  described,  is  the  more  virulent  and  sometimes 
dangerous  Staphylococcus  pyogenes  aureus,  or  "  golden  staphylococ- 
cus,"  first  observed  by  Ogston  and  cultivated  by  Rosenbach.  As 
the  morphology  and  cultural  characteristics  of  this  organism  are 
identical  with  those  of  the  preceding  species,  it  seems  convenient  to 
describe  them  together,  pointing  out  such  minor  differences  as  occur. 
In  doing  this,  however,  it  must  not  be  forgotten  that,  although 
Staphylococcus  albus  was  first  mentioned,  Staphylococcus  aureus 
is  the  more  common  organism  of  suppuration. 

STAPHYLOCOCCI  PYOGENES  AUREUS  ET  ALBUS 

Distribution. — The  cocci  are  not  widely  distributed  in  nature, 
seeming  not  to  find  a  purely  saprophy  tic  existence  satisfactory .  They 


\ 


Fig.  106. — Staphylococcus  pyogenes  aureus  (Gunther). 

occur,  however,  upon  man  and  the  lower  animals,  and  can  occasionally 
be  found  in  the  dusts  of  houses  and  hospitals — especially  in  the 
surgical  wards — if  proper  precautions  are  not  exercised.  They  are 
common  upon  the  skin,  in  the  nose,  mouth,  eyes,  and  ears  of  man; 
they  are  nearly  always  present  beneath  the  finger-nails,  and  some- 
times occur  in  the  feces,  especially  of  children. 

Staphylococci  are  the  most  common  micro-organisms  in  some 
acne  pustules,  in  furuncles,  in  carbuncles,  in  superficial  and  deep 
abscesses,  and  in  the  ordinary  run  of  surgical  injections.  So  com- 
mon are  they  that  one  should  never  be  satisfied  that  he  has  exhausted 

*  "  Mikroorganismen  bei  Wundinfektionskrankheiten  des  Menschen, "  Wies- 
baden, 1884. 


Staphylococcus  Pyogenes  Aureus  et  Albus  303 

the  etiological  possibilities  of  the  case  through  their  demonstration. 
He  should  always  seek  for  less  evident  though  sometimes  far  more 
important  organisms.  In  the  absence  of  such,  and  in  their  absence 
only,  should  the  case  be  referred  to  staphylococci. 

Morphology. — The  cocci  are  small  spheres  measuring  about  0.7- 
i.o  \i  in  diameter.  There  is  no  definite  grouping  in  either  liquid 
or  solid  cultures.  It  is  only  in  pus  or  in  the  organs  or  tissues  of  dis- 
eased animals  that  one  can  say  that  a  true  Staphylococcus  (bunch 
of  grapes)  grouping  occurs. 

The  organisms  are  not  motile  and  have  no  flagella.  They  do  not 
form  spores. 

Staining. — They  stain  easily  and  brilliantly  with  aqueous  solutions 
of  the  anilin  dyes  and  by  Gram's  method. 


Fig.  107. — Staphylococcus  pyogenes  aureus.     Colony  two  days  old,  seen  upon 
an  agar-agar  plate.      X  40  (Heim). 

Isolation. — Staphylococci  are  easy  organisms  to  isolate,  and  can 
be  secured  by  plating  out  a  drop  of  pus  in  gelatin  or  in  agar-agar. 

The  colonies  of  Staphylococcus  aureus  differ  considerably  in 
color,  some  being  much  paler  than  others. 

Cultivation. — The  staphylococci  grow  well  upon  all  the  standard 
culture-media  either  in  the  presence  or  in  the  absence  of  oxygen  at 
temperatures  above  i8°C.,  the  most  rapid  development  being  at 
about  37°C. 

Colonies. — Upon  the  surface  of  gelatin  plates  the  colonies  appear 
as  small  whitish  points,  after  from  twenty-four  to  forty-eight  hours, 
rapidly  extending  to  the  surface  and  causing  extensive  liquefaction 
of  the  medium.  The  formation  of  the  yellow  pigment  can  be  best 
observed  near  the  center  of  the  colonies.  Under  the  microscope  the 
colonies  appear  as  round  disks  with  circumscribed,  smooth  edges. 
They  are  distinctly  granular  and  dark  brown.  When  the  colonies 
are  grown  upon  agar-agar  plates,  the  formation  of  the  pigment  is 
more  distinct. 


3°4 


Suppuration 


Gelatin  Punctures. — In  gelatin  the  growth  occurs  along  the  whole 
length  of  the  puncture,  causing  an  extensive  liquefaction  of  the 
medium  in  the  form  of  a  long,  narrow,  blunt-pointed,  inverted  cone, 
sometimes  described  as  being  like  a  stocking,  full  of  clouded  liquid, 
at  the  apex  of  which  a  collection  of  golden  or  orange-yellow  precipitate 
is  always  present  in  Staphylococcus  aureus.  It  is  this  precipitate 
in  particular  that  gives  the  organism  its  name,  "golden  staphylo- 
coccus." 

Agar-Agar. — The   growth   of    the   golden 
Staphylococcus  upon  agar-agar  is  subject  to  con- 
siderable variation  in  the  quantity  of  pigment 
produced.     Sometimes,    perhaps    rarely,    it    is 
BpPP  golden;  more  commonly  it  is  yellow,  often  cream 

color.  Along  the  whole  line  of  inoculation  a 
moist,  shining,  usually  well-circumscribed 
growth  occurs.  When  the  development  occurs 
rapidly,  as  in  the  incubator,  it  exceeds  the 
rapidity  of  color  production,  so  that  the  center 
of  the  growth  is  distinctly  colored,  the  edges 
remaining  white. 

Potato. — Upon  potato  the  growth  is  luxu- 
riant, Staphylococcus  aureus  producing  an 
orange-yellow  coating  over  a  large  part  of  the 
surface.  The  potato  cultures  may  give  off  a 
sour  odor. 

Bouillon. — When  grown  in  bouillon  the  organ- 
ism causes  a  diffuse  cloudiness,  with  a  small 
quantity  of  slightly  yellowish  sediment.  The 
reaction  of  the  medium  becomes  increasingly 
acid.  Nitrates  are  reduced  to  nitrites. 

Milk. — In  milk,  coagulation  takes  place  in 
about  eight  days,  and  is  followed  by  gradual 
digestion  of  the  casein.  In  litmus  milk  slow 
acid  production  is  observed. 
Thermal  Death  Point. — Staphylococci  are  usually  quite  suscep- 
tible to  the  effect  of  heat,  though  their  resistance  is  not  uniform. 
Sternberg  found  them  destroyed  by  an  exposure  to  62°C.  for  ten 
minutes,  and  to  8o°C.  for  one  and  a  half  minutes,  but  three  cultures 
studied  by  von  Lingelsheim  were  not  killed  by  an  exposure  to  6o°C. 
for  an  hour,  and  one  culture  studied  by  him  endured  an  exposure 
to  8o°C.  for  ten  minutes. 

Metabolic  Products. — Staphylococci  can  make  use  of  free  or 
combined  oxygen,  hence  are  aerobic  or  anaerobic.  In  liberating 
combined  oxygen,  no  gas  is  generated  in  any  culture  medium.  They 
produce  ferments  by  which  gelatin  is  liquefied,  milk  coagulated  and 
digested,  blood-serum  digested  and  slowly  liquefied.  A  yellow 
pigment  is  produced.  Nitrates  are  reduced  to  nitrites  in  cultures 


Fig.  108.— Staph- 
ylococcus pyogenes 
aureus.  Puncture 
culture  three  days 
old  in  gelatin  (Fran- 
kel  and  Pfeiffer). 


Staphylococcus  Pyogenes  Aureus  et  Albus  305 

kept  for  three  days  at  37°C.  Staphylococci  are  capable  of  producing 
fatty  acids  from  sugars,  hence  acidity  develops  in  media  containing 
lactose,  maltose,  mannite  and  glycerin.  The  acids  most  commonly 
produced  are  acetic,  valerianic,  butyric  and  propionic. 

Toxic  Products. — Leber  seems  to  have  first  conceived  of  suppura- 
tion as  a  toxic  process  depending  upon  the  soluble  products  of 
parasitic  fungi,  and  in  1888,  through  the  action  of  alcohol  upon 
Staphylococci,  prepared  an  acicular  crystalline  body  soluble  in 
alcohol  and  ether,  but  slightly  soluble  in  water,  to  which  he  gave 
the  name  phlogosin. 

Mannatti  found  that  pus  has  substantially  the  same  toxic  prop- 
erties as  sterilized  cultures  of  the  Staphylococcus;  that  repeated  in- 
jections of  sterilized  pus  induce  chronic  intoxication  and  marasmus; 
that  injection  of  sterilized  pus  under  the  skin  causes  a  grave  form  of 
poisoning;  and  that  the  symptoms  and  pathologic  lesions  caused  by 
these  injections  correspond  with  those  observed  in  men  suffering 
from  chronic  suppuration. 

Van  de  Velde*  found  that  the  Staphylococcus  has  some  metabolic 
products  destructive  to  the  leukocytes,  which  he  has  called  leuko- 
cidin.  This  poison  causes  the  cells  to  cease  ameboid  movement, 
become  spheric,  and  gradually  to  lose  their  granules,  until  they  finally 
appear  like  empty  sacs  containing  shadow  nuclei,  which  eventually 
disappear.  The  leukolysis  occurs  in  about  two  minutes.  These 
observations  have  been  abundantly  confirmed.  Kraussf  first  ob- 
served that  certain  products  of  the  Staphylococcus  were  hemolytic 
and  destroyed  red  blood-corpuscles.  This  hemolysin  has  been 
carefully  studied  by  Neisser  and  Wechsberg,J  by  whom  it  was 
called  staphylolysin. 

Durme§  found  staphylolysin  produced  most  abundantly  by 
virulent  Staphylococci. 

Ribbert||  found  that  both  sterilized  and  unsterilized  cultures  when 
intravenously  injected  into  animals  produced  definite  changes  in 
the  heart,  kidneys,  lungs,  spleen,  and  bone-marrow,  and  attributed 
the  action  to  the  toxin. 

Morse**  found  that  the  toxic  products  of  Staphylococcus  aureus 
were  capable  of  occasioning  interstitial  nephritis. 

The  Staphylococci  form  very  little  extracellular  toxin,  as  filtered 
cultures  provoke  little  local  or  general  reaction  in  animals,  even 
when  the  Staphylococcus  is  highly  virulent. 

To  secure  the  endo-toxin,  masses  of  culture,  prepared  as  described 
in  the  section  upon  "Bacterio- vaccines,"  are  ground  in  a  mortar,  or 

*  'La  Cellule,"  1896,  xi,  p.  349. 

;  Wiener,  klin.  Wochenschrift,"  1900. 

:Zeitschrift  fur  Hygiene/'  1911,  xxxvi,  p.  330. 

Hyg.  Rundschau,"  1903,  Heft  2,  p.  66. 

:  Die  pathologische  Anatomic  und  die  Heilung  der  durch  den  Staphylococcus 
pyogenes  aureus  hervorgerufenen  Erkrankungeri." 

**  "Journal  of  Experimental  Medicine,"  1896,  vol.  i,  p.  613. 


306  Suppuration 

frozen  by  liquid  air  and  then  ground,  or  the  culture  masses  are  treated 
by  dilute  acids  and  alkalies  according  to  Vaughan,  or  the  culture 
masses  are  permitted  to  undergo  autolysis  in  physiological  salt 
solution  or  in  diluted  serum  containing  amboceptor  and  complement 
(see  Bacteriolysis). 

Pathogenesis. — The  virulent  Staphylococcus  aureus  is  a  danger- 
ous and  sometimes  a  deadly  organism.  Its  virulence  is,  however, 
very  variable  both  for  the  lower  animals  and  for  man.  The  most 
susceptible  laboratory  animal  is  the  rabbit.  Guinea-pigs,  rats,  mice, 
dogs  and  cats  are  much  less  susceptible. 

The  classical  test  for  virulence  is  to  inject  J{0  cc-  °f  a  twenty- 
four  hour  old  bouillon  culture  into  the  ear  vein  of  a  middle-sized 
rabbit.  If  of  the  ordinary  virulence,  the  organism  should  kill  the 
rabbit  in  from  four  to  eight  days,  during  which  time  the  animal 
suffers  from  fever  and  wasting.  Highly  virulent  cultures  kill  the 
animal  in  from  one  to  two  days. 

The  effects  produced  by  different  methods  of  inoculation  are  marked. 
Thus,  if  a  few  drops  of  a  virulent  culture  be  injected  beneath  the  skin 
of  a  rabbit,  there  is  a  local  reaction,  an  abscess  forms,  the  temperature 
rises  and  the  animal  is  ill.  In  a  few  days  the  abscess  points  and 
empties,  the  temperature  returns  to  the  normal  and  the  animal 
recovers.  In  exceptional  cases  a  generalized  injection  occurs  and 
the  rabbit  dies. 

If  the  injection  be  made  into  the  peritoneal  cavity,  pleural  cavity  or 
into  a  joint,  there  is  primarily  a  localized  suppuration,  peritonitis, 
pleuritis  or  arthritis,  which  is  usually  followed  in  a  day  or  two  by 
generalized  infection  and  death. 

Intravenous  injections  are  immediately  followed  by  rise  of  tem- 
perature, and  the  occurrence  of  multiple  widespread  foci  of  coloniza- 
tion with  minute  abscesses  in  many  of  the  organs.  The  heart  is 
sometimes  the  seat  of  purulent  myocarditis,  less  frequently  of  septic 
endocarditis.  The  kidneys  show  minute  abscesses,  with  aggregations 
of  cocci  in  the  glomeruli  and  in  the  tubules. 

When  the  cocci  enter  human  beings  subcutaneously,  furuncles, 
carbuncles  and  abscesses  commonly  result,  according  to  the  virulence 
of  the  organism  and  the  resisting  power  of  the  individual.  Garre* 
applied  the  organism  in  pure  culture  to  the  uninjured  skin  of  his  arm, 
and  in  four  days  developed  a  large  carbuncle,  with  a  surrounding 
zone  of  furuncles.  Bockhartf  suspended  a  small  portion  of  an  agar- 
agar  culture  in  salt  solution,  and  scratched  it  gently  into  the  deeper 
layer  of  the  skin  with  his  finger-nail;  a  furuncle  developed.  Bumm 
injected  the  coccus  suspended  in  salt  solution  beneath  his  skin  and 
that  of  several  other  persons,  and  produced  an  abscess  in  every  case. 
When  conditions  of  invasion  are  most  favorable,  fatal  generalization 
of  the  organisms  may  occur.  In  such  cases  they  may  be  cultivated 

-.*  " Fortschritte  der  Med.,"  1885,  No.  6. 
t  "  Monatschrif t  fur  prakt.  Dermatologie,"  1887,  iv,  No.  10. 


Staphylococcus  Pyogenes  Aureus  et  Albus  307 

from  the  streaming  blood,  though  the  greater  number  collect  in, 
and  frequently  obstruct,  the  capillaries.  In  the  lungs  and  spleen, 
and  still  more  frequently  in  the  kidneys,  infarcts  are  formed  by  the 
bacterial  emboli.  The  Malpighian  tufts  of  the  kidneys  are  sometimes 
full  of  cocci,  and  become  the  centers  of  small  abscesses. 

It  enters  the  human  system  through  scratches,  punctures,  or 
abrasions,  and  when  virulent  usually  occasions  an  abscess. 

Staphylococcus  aureus  is  not  only  found  in  the  great  majority 
of  furuncles,  carbuncles,  abscesses,  and  other  inflammatory  dis- 
eases of  the  surface  of  the  body,  but  also  plays  an  important  role  in 
a  number  of  deeply  seated  diseases.  Becker  and  others  obtained  it 
from  the  pus  of  osteomyelitis,  demonstrating  that  if,  after  fracturing 
or  crushing  a  bone,  the  Staphylococcus  be  injected  into  the  circu- 
lation, osteomyelitis  may  occur.  Numerous  observers  have  demon- 
strated its  presence  in  ulcerative  endocarditis.  Rodet  has  been 
able  to  produce  osteomyelitis  without  previous  injury  to  the  bones; 
Rosenbach  was  able  to  produce  ulcerative  endocarditis  by  injecting 
some  of  the  staphylococci  into  the  circulation  in  animals  whose 
cardiac  valves  had  been  injured  by  a  sound  passed  into  the  carotid 
artery;  and  Ribbert  has  shown  that  the  injection  of  cultures  of  the 
organism  may  cause  valvular  lesions  without  preceding  injury. 

Virulence. — Experiments  have  shown  that  both  Staphylococci 
aureus  and  albus  exist  in  attenuated  and  virulent  forms,  and  there 
is  every  reason  to  believe  that  in  the  majority  of  instances  they  in- 
habit the  surface  of  the  body  in  a  feebly  virulent  condition. 

Agglutination. — Kolle  and  Otto*  have  found  that  -immune  anti- 
staphylococcic  serums  agglutinate  the  staphylococci.  The  reaction 
is  not  specific  and  is  peculiar.  All  pathogenic  staphylococci  are 
agglutinated;  non-pathogenic  cocci  are  not  agglutinated.  The 
reaction  cannot,  therefore,  be  used  for  specific  differentiation. 

Specific  Therapy. — The  treatment  of  Staphylococcus  infections 
with  immune  serum  has  not  met  with  encouraging  success.  Vi- 
querat,f  Deny  sand  van  de  Velde,{  and  Neisser  and  Wechsberg§  and 
others  have  experimented  in  this  direction,  but  the  literature 
contains  very  little  evidence  that  beneficial  results  have  followed  the 
employment  of  antistaphylococcus  serums. 

Bacterio-vaccination. — Although  specific  serums  have  failed,  a 
promising  form  of  specific  treatment  for  subacute  and  chronic 
staphylococcic  infections  has  been  introduced  by  A.  E.  Wright, || 
who  first  isolates  from  the  lesion  the  particular  strain  of  staph- 
ylococci by  which  it  is  caused,  cultivates  this  artificially,  suspends 
the  organisms  in  an  indifferent  fluid,  of  which  a  given  quantity  con- 
tains a  known  (counted)  number,  kills  the  organisms  by  heating  them 

*  "Zeitschrift  fur  Hygiene,"  etc.,  1902,  XLI. 
t  Ibid.,  xvm,  1894,  p.  483. 
:  "La  Cellule,"  1895,  xi. 
§  "Zeitschrift  fur  Hygiene,"  1901,  xxxn. 
||  "Lancet,"  March  29,  1902,  p.  874;  "Brit.  Med.  Jour.,"  May  9,  1903,  p.  1069. 


308  Suppuration 

for  an  hour  at  6o°C.,  and  then  uses  them  by  subcutaneous  injection 
for  producing  increased  resistance  on  the  part  of  the  patient.  (See 
"  Bacterio- vaccination. ") 

The  treatment  is  controlled  by  studying  the  " opsonic  index" 
(q.v.),  the  objects  being  the  avoidance  of  the  "negative  phase"  or 
condition  of  diminished  resistance,  and  the  progressive  establish- 
ment of  the  positive  phase  or  stage  of  increased  resistance.  As  the 
resistance  increases  the  patient  rapidly  improves,  and  many  cases 
of  obstinate  acne,  furunculosis,  and  other  pyogenic  infections  have 
quickly  recovered  under  this  treatment. 

STAPHYLOCOCCUS  CITREUS  (PASSET) 

An  organism  similar  in  many  respects  to  the  preceding,  except 
that  its  growth  on  agar-agar  and  potato  is  of  a  brilliant  lemon- 
yellow  color  and  its  pathogenicity  for  animals  doubtful,  is  Staph- 
ylococcus  citreus  of  Passet.*  As  it  is  not  common  and  is  doubtfully 
pathogenic,  it  is  of  much  less  importance  than  the  previously 
described  organisms. 

STREPTOCOCCUS  PYOGENES  (ROSENBACH) 

General  Characteristics. — The  streptococcus  is  a  non-motile,  non-flagellate, 
non-sporogenous,  non-liquefying,  non-chromogenic,  aerobic  and  optionally 
anaerobic,  spheric  organism,  infectious  for  man  and  the  lower  animals.  It 
stains  by  ordinary  methods  and  by  Gram's  method. 

Streptococci  were  probably  first  seen  by  Pasteur  and  Doleris  in 
the  blood  of  women  suffering  from  puerperal  infection,  and  by 
Kochf  in  1878.  In  1881  OgstonJ  called  attention  to  the  fact  that 
two  distinct  kinds  of  cocci  were  to  be  found  in  pus,  mentioning  both 
staphylococci  and  streptococci.  The  beginning  of  real  knowledge 
of  the  streptococci,  however,  dates  from  the  time  of  their  isolation 
and  cultivation  by  Fehleisen§  and  of  Rosenbach,||  from  18  of  33 
suppurative  lesions,  fifteen  times  alone  and  five  times  in  association 
with  Staphylococcus  aureus. 

Distribution. — Streptococci  are  parasitic  pathogenic  organisms, 
not  known  apart  from  human  and  animal  hosts.  They  seem  to  occur 
not  infrequently,  in  health,  upon  the  surf  ace  of  the  body,  in  its  various 
openings  and  in  the  alimentary  canal.  Such  organisms  are  to  be 
regarded  as  potentially  virulent  and  pathogenic  in  all  cases. 

Streptococci    have    been   the    subject   of    extensive   systematic 

*  "  Untersuchungen  iiber  die  Aetiologie  der  eitrigen  Phlegmone  des  Menschen," 
Berlin,  1885,  p.  p. 

t  "Untersuchungen  iiber  die  Aetiologie  der  Wundinfektionskrankheiten," 
Leipzig,  Vogel,  1878. 

t  "British  Med.  Jour.,"  March,  1881,  p.  369. 

§  "Aetiologie  des  Erysipels,"  Berlin,  Fischer,  1883. 

||  "  Mikroorganismen  bei  Wundinfektionskrankheiten  des  Menschen,"  1884, 
p.  22. 


Streptococcus  Pyogenes  309 

study  because  of  still  existing  uncertainty  as  to  whether  there  is  a 
single  species  or  whether  there  are  various  species,  but  opinion,  at 
present,  seems  in  favor  of  the  opinion  that  there  is  but  one  strep- 
tococcus whose  various  manifestations  depend  upon  its  virulence, 
upon  the  resistance  of  the  host,  upon  its  avenue  of  entrance,  and 
the  associated  micro-organisms  with  which  it  happens  to  engage. 

Streptococci  may  be  primary  pathogenic  agents,  or  they  may  be 
secondary  agents  whose  activities  complicate,  modify  and  sometimes 
outweigh  in  importance  those  of  the  primary  agents. 

They  are  the  primary  infecting  agents  in  many  inflammatory, 
purulent  and  septicemic  disturbances — erysipelas,  cellulitis,  phleg- 
mons, osteomyelitis,  puerperal  infection,  pseudo-membranous  angina, 
phlebitis,  salpingitis,  meningitis,  endocarditis,  etc. 


Fig.   109. — Streptococcus  pyogenes,  from  the  pus  taken  from  an  abscess. 
X  1000  (Frankel  and  Pfeiffer). 

Berson  points  out  that  they  are  secondary  agents  of  importance 
in  all  pathological  conditions  of  the  throat  of  whatever  nature. 

Hektoen  found  them  to  be  the  most  frequent  complicating  organism 
in  scarlatina  and  Councilman  the  most  frequent  complicating 
organism  in  variola. 

The  suppurative  conditions  for  which  streptococci  are  held  to  be 
responsible,  differ  from  those  caused  by  staphylococci  in  being  more 
rapidly  spreading,  more  locally  destructive,  and  more  prone  to 
generalized  infection  or  septicemia. 

Morphology. — The  organisms  are  spheric,  of  variable  size  (0.4-1  A* 
in  diameter),  and  are  constantly  associated  in  pairs  or  in  chains  of 
from  four  to  twenty  or  more  individuals.  Special  varieties,  known 
as  Streptococcus  longus  (chains  of  more  than  one  hundred  members) 


3io  Suppuration 

and  Streptococcus  brevis  (chains  of  from  four  to  ten),  have  been 
described  by  v.  Lingelsheim,*  but  do  not  hold  as  separate  species. 

The  streptococcus  is  not  motile  and  does  not  form  spores. 

Staining. — The  organisms  stain  well  with  ordinary  aqueous 
solutions  of  anilin  dyes  and  by  Gram's  method. 

Isolation. — The  streptococcus  can  be  isolated  from  pus  contain- 
ing it  by  plating  or  by  the  inoculation  of  a  mouse  or  rabbit,  from 
whose  blood  it  may  easily  be  secured  after  death. 

Cultivation. — The  organism  grows  at  both  the  room  temperature 
and  that  of  incubation,  its  best  and  most  rapid  development  being 
at  about  37°C. 

.  Colonies. — Upon  gelatin  plates  very  small,  colorless,  translucent 
colonies  appear  in  from  twenty-four  to  forty-eight  hours.  When 
superficial,  they  spread  out  to  form  flat  disks  about  0.5  mm.  in 
diameter.  The  microscope  shows  them  to  be  irregula  nd  granular, 
to  have  a  slightly  yellowish  color  by  transmitted  lig  ,  and  to  have 
a  frayed-out  appearance  around  the  edges,  due  to  projecting  chains 
of  the  cocci.  No  liquefaction  of  the  gelatin  occurs. 

Gelatin  Punctures. — In  gelatin  puncture  cultures  no  liquefaction 
is  observed.  The  minute  spheric  colonies  grow  along  the  whole 
length  of  the  puncture  and  form  a  slightly  opaque  granular  line. 


Fig.  no. — Streptococcus  colonies  on  serum  agar  (From  Hiss  and  Zinsser, 
"Text-Book  of  Bacteriology,"  D.  Appleton  &  Co.,  Publishers). 

Agar-agar. — Upon  agar-agar  a  delicate  transparent  growth  de- 
velops slowly  along  the  line  of  inoculation.  It  consists  of  small, 
colorless,  or  slightly  grayish  transparent  colonies  which  do  not  readily 
coalesce. 

Blood-serum. — The  growth  upon  blood-serum  resembles  that  upon 
agar-agar.  The  colonies  are  small,  white,  discrete,  and  do  not  affect 
the  medium. 

Potato. — The  streptococcus  does  not  seem  to  grow  well  upon 
potato,  the  colonies  being  invisible. 

Bouillon. — In  bouillon  the  cocci  develop  slowly,  seeming  to  prefer 
a  neutral  or  feebly  alkaline  reaction.  The  medium  remains  clear, 

*  "Zeitschrift  fur  Hygiene,"  1891,  Bd.  x,  p.  331;  1892,  xn,  p.  308. 


Streptococcus  Pyogenes  311 

while  numerous  small  flocculi  are  suspended  in  it,  sometimes  ad- 
hering to  the  sides  of  the  tube,  sometimes  forming  a  sediment. 
When  the  flocculi  formation  is  distinct,  the  name  Streptococcus  con- 
glomeratus  (Kurth)  is  sometimes  given  to  the  organism;  when  the 
medium  is  diffusely  clouded,  it  is  called  Streptococcus  di/usus. 

In  mixtures  of  bouillon  and  blood-serum  or  ascitic  fluid  the  strep- 
tococcus grows  more  luxuriantly,  especially  at  incubation  tempera- 
tures, distinctly  clouding  the  liquid.  As  the  lactic  acid  which  is 
rapidly  formed  inhibits  the  growth  of  the  cocci,  Hiss  recommends* 
that  instead  of  eliminating  the  'sugars  in  the  broth,  upon  which  the 
streptococci  are  nourished,  i  per  cent,  of  sterile  powdered  CaCOs 
be  added  to  the  culture-media.  This  neutralizes  the  acid  as  rapidly 
as  it  is  formed.  It  also  maintains  the  life  of  the  culture  for  a  long 
time. 

Milk. — TV  >rganism  seems  to  grow  well  in  milk,  which  is  coagu- 
lated and  d%.i  ^ed. 

Reaction.— "ifrie  streptococcus  is  sensitive  to  acids,  and  can  only 
grow  well  in  media  with  a  slightly  alkaline  reaction.  All  strepto- 
cocci produce  acids  and  eventually  acidulate  the  media,  thus  check- 
ing their  further  development. 

Vital  Resistance. — The  optimum  temperature  appears  to  be  in  the 
neighborhood  of  37°C.  It  grows  well  between  25°  and  4O°C.,  above 
4o.5°C.  the  growth  is  slowed.  The  thermal  death  point  is  low. 
Sternberg  found  that  the  streptococci  succumb  at  temperatures  of 
52°  to  54°C.  if  maintained  for  ten  minutes.  Their  vitality  in  culture 
is  slight,  and  unless  frequently  transplanted  they  die.  Bouillon 
cultures  usually  die  in  from  five  to  ten  days.  On  solid  media  they 
seem  to  retain  their  vegetative  and  pathogenic  powers  much  longer, 
especially  if  kept  cool  and  cultivated  beneath  the  surface  of  the 
medium  in  a  deep  puncture.  They  resist  drying  fairly  well. 

Differential  Features. — It  is  not  always  easy  to  differentiate 
Streptococcus  pyogenes  from  other  less  important  forms  of  strep- 
tococci and  from  the  pneumococcus.  One  of  the  best  methods  is 
by  the  employment  of  Uood-agar  plates,  suggested  by  Schottmuller.t 
Such  plates  are  easily  prepared  by  melting  ordinary  culture  agar- 
agar,  cooling  to  about  45°C.,  and  then  adding  about  0.5  cc.  of  de- 
fibrinated  human  or  rabbit's  blood  to  the  tube.  The  blood  is  first 
thoroughly  mixed  with  the  agar,  then  the  tube  inoculated,  and  poured 
into  a  Petri  dish.  As  the  Streptococcus  pyogenes  grows,  it  produces 
a  hemolytic  substance  that  destroys  the  blood-corpuscles  in  the 
vicinity  of  the  colony,  thus  surrounding  each  by  a  clear,  pale  halo 
that  contrasts  with  the  red  agar.  The  colonies  themselves  appear 
gray. 

The  test  is  not  specific,  and  Ruediger %  points  out  that  the  diph- 

*  "Text-book  of  Bacteriology,"  p.  338. 

t  "Munch,  med.  Wochenschrift,"  1903,  L,  p.  909. 

j  "Jour.  Amer.  Med.  Assoc.,"  1906,  XLVII,  p.  1171. 


312  Suppuration 

theria  and  pseudodiphtheria  bacilli  also  produce  hemolyzing  sub- 
stance, so  that  the  test  cannot  be  used  for  the  immediate  separation 
of  streptococci  from  other  bacteria  in  cultures  from  the  throat. 
Colonies  of  the  pneumococcus  usually  appear  green  and  without 
hemolysis,  but  Ruediger  finds  that  they  also  sometimes  cause 
solution  of  the  hemoglobin.  The  streptococci  whose  colonies  are 
green  and  without  hemolysis  are  called  Streptococcus  mridans  by 
Schottmiiller.  They  were  at  first  regarded  as  practically  non- 
pathogenic,  but  it  is  now  known  that  they  cause  endocarditis  in 
rabbits  and  it  is  thought  that  they  may  do  so  in  man. 

Pathogenesis.  —  The  streptococcus  has  been  found  in  erysipelas, 
malignant  endocarditis,  periostitis,  otitis,  meningitis,  empyema, 
pneumonia,  lymphangitis,  phlegmons,  sepsis,  puerperal  endo- 
metritis,  and  many  other  forms  of  inflammation  and  septic  infection. 
In  man  it  is  usually  associated  with  active  suppuration  and  sepsis. 

The  relation  of  the  streptococcus  to  diphtheria  is  of  interest,  for, 
though  in  all  probability  the  great  majority  of  cases  of  pseudo- 
membranous  angina  are  caused  by  the  Klebs-Loffler  bacillus,  yet  a 
number  are  met  with  in  which,  as  in  Prudden's  24  cases,  no  diphtheria 
bacilli  can  be  found,  but  which  seem  to  be  caused  by  the  strepto- 
coccus alone. 

There  are  few  clinical  differences  between  the  throat  lesions  pro- 
duced by  the  two  organisms,  and  the  only  positive  method  of  dif- 
ferentiating the  one  from  the  other  is  by  means  of  a  careful  bacterio- 
logic  examination.  Such  an  examination  should  always  be  made, 
as  it  has  much  weight  in  connection  with  the  treatment;  in  strepto- 
coccus angina  no  benefit  can  be  expected  from  the  administration  of 
diphtheria  antitoxic  serum. 

Hirsh*  has  shown  that  streptococci  are  by  no  means  rare  in  the 
intestines  of  infants,  where  they  may  occasion  enteritis.  In  such 
cases  the  organisms  are  found  in  large  numbers  in  the  stomach  and 
in  the  stools,  and  late  in  the  course  of  the  disease  in  the  blood  and 
urine  of  the  child.  They  also  occur  in  all  of  the  internal  organs  of 
the  cadaver. 

The  intestinal  streptococci  are  often  Gram-negative,  when  they 
are  usually  non-virulent. 

Libmanf  has  reported  2  carefully  studied  cases  of  streptococcic 
enteritis. 

Flexner,f  in  a  larger  series  of  autopsies,  found  the  bodies  in- 
vaded by  numerous  micro-organisms,  causing  what  he  has  called 
"  terminal  infections,"  and  hastening  the  fatal  issue.  Of  793 
autopsies  at  the  Johns  Hopkins  Hospital,  255  upon  cases  dying  of 
chronic  heart  or  kidney  diseases,  or  both,  were  sufficiently  well  studied 
bacteriologically,  to  meet  the  requirements  of  a  statistical  inquiry. 

!  "Centralbl.  f.  Bakt.  u.  Parasit.,"  Bd.  xxn,  Nos.  14  and  15,  p.  369. 
t  "  Centralbl. 'f.  Bakt.  u.  Parasit.,"  Bd.  xxn,  Nos.  14  and  15,  p.  376. 
t  "Journal  of  Experimental  Medicine,"  1896,  vol.  I,  No.  3. 


Streptococcus  Pyogenes  313 

Tuberculous  infections  were  not  included.  Of  the  255  cases,  213 
gave  positive  bacteriologic  results.  "The  micro-organisms  causing 
the  infections,  38  in  all,  were  Streptococcus  pyogenes,  16  cases; 
Staphylococcus  pyogenes  aureus,  4  cases;  Micrococcus  lanceolatus, 
6  cases;  gas  bacillus  (Bacillus  aerogenes  capsulatus),  three  times 
alone  and  twice  combined  with  B.  coli  communis;  the  gonococcus, 
anthrax  bacillus;  B.  proteus,  the  last  combined  with  B.  coli;  B. 
coli  alone;  a  peculiar  capsulated  bacillus,  and  an  unidentified 
coccus." 

It  is  interesting  to  observe  in  how  many  cases  the  streptococcus 
was  present.  All  the  streptococci  found  may  not  have  been  Strepto- 
coccus pyogenes,  but  for  convenience  in  his  statistics  they  were  re- 
garded as  such. 

The  presence  of  streptococci  in  the  blood  in  scarlatina  has  been 
observed  in  30  cases  by  Crooke,  by  Frankel  and  Trendenburg, 
Raskin,  Leubarth,  Kurth,  and  Babes.  In  n  cases  of  scarlatina 
studied  by  Wright*  a  general  streptococcus  infection  occurred  in 
4,  a  penumococcus  infection  in  i,  and  a  mixed  infection  of  pyogenic 
cocci  in  i. 

Lemoinef  found  streptococci  in  the  blood  during  life  in  2  out  of 
33  cases  of  scarlet  fever  studied.  PearceJ  studied  1 7  cases  of  scarla- 
tina and  found  streptococci  in  the  heart's  blood  and  liver  in  4,  in 
the  spleen  in  2,  in  the  kidney  in  5  cases.  In  2  of  the  cases  Staphy- 
lococcus pyogenes  aureus  was  associated  with  the  streptococcus. 

The  streptococcus  is  the  most  common  organism  found  in  the 
suppurative  sequelae  of  scarlatina,  frequently  occurring  alone; 
sometimes  with  the  staphylococci ;  sometimes  with  the  pneumococci. 

Virulence. — Streptococci  isolated  from  human  beings  vary  greatly 
in  pathogenic  action  upon  the  laboratory  experiment  animals.  In 
many  cases,  although  they  have  induced  a  fatal  illness  in  human 
beings,  they  are  without  effect  upon  the  lower  animals;  in  other 
cases,  although  from  a  more  simple  lesion  that  recovered,  they  are 
extremely  fatal  for  the  most  susceptible  animals,  rabbits  and  mice. 
Rats  sometimes  become  ill  when  injected  with  virulent  cultures  in 
large  doses,  but  usually  recover.  Guinea-pigs,  cats,  and  dogs  are 
but  slightly  susceptible  even  when  the  cultures  are  virulent.  Large 
animals,  like  sheep,  goats,  cattle,  and  horses,  react  very  slightly 
to  large  doses,  but  sometimes  suffer  from  abscesses  at  the  seat  of 
injection.  Mice  die  in  from  one  to  four  days  from  general  infec- 
tion. If  the  organisms  are  less  virulent,  they  die  in  from  four  to 
six  days  with  edema  and  abscess  formation  at  the  site  of  inocula- 
tion, and  subsequent  invasion  of  the  body.  All  streptococci  seem 
to  be  most  pathogenic  for  that  species  of  animal  from  which  they 
have  been  isolated. 

*  "Boston  Med.  and  Surg.  Jour.,  "March  21,  1895. 

t  "Bull,  et  Mem.  Soc.  d'H6p.  de  Paris,"  1896,  3  s.,  xm. 

j  "Jour.  Boston  Soc.  of  Med.  Sci.,"  March,  1898. 


314  Suppuration 

If  the  ear  of  a  rabbit  be  carefully  scarified,  and  cutaneously  in- 
oculated with  a  small  quantity  of  a  pure  culture,  local  erysipelas 
usually  results,  the  disturbance  passing  away  in  a  few  days  and  the 
animal  recovering.  If,  however,  the  streptococcus  be  highly  viru- 
lent, the  rabbit  may  die  of  general  septicemia  in  from  twenty- 
four  hours  to  six  days.  The  cocci  may  then  be  found  in  large 
numbers  in  the  heart's  blood  and  in  the  organs.  In  less  virulent 
cases  minute  disseminated  pyemic  abscesses  are  sometimes  found. 

When  mildly  virulent  cultures  of  the  variety  called  Streptococcus 
viridans  are  intravenously  injected  into  rabbits,  some  time  elapses 
before  much  disturbance  is  noted,  then  the  animal  becomes  ill  and 
eventually  dies  of  cardiac  disease.  Verrucose  endocarditis  with 
marked  calcification  of  the  mitral  valve,  with  secondary  metastatic 
subacute  glomerulonephritis  was  observed  in  those  cases  which  were 
carefully  studied  by  Libman.* 

According  to  Marmorek,f  the  virulence  of  the  streptococcus 
can  be  increased  to  a  remarkable  degree  by  rapid  passage  through 
rabbits,  and  maintained  by  the  use  of  a  culture-medium  consisting 
of  3  parts  of  human  blood-serum  and  i  of  bouillon.  The  blood  of 
the  ass  or  ascitic  or  pleuritic  exudates  may  be  used  instead  of  the 
human  blood-serum  if  the  latter  be  unobtainable.  By  these  means 
he  succeeded  in  intensifying  the  virulence  of  a  culture  to  such  a 
degree  that  one  hundred-thousand  millionth  (un  cent  milliardieme) 
of  a  cubic  centimeter  injected  into  the  ear  vein  was  fatal. 

Petruschky}  found  the  virulence  of  the  culture  to  be  well  re- 
tained when  the  organisms  were  planted  in  gelatin,  transplanted 
every  five  days,  and  when  grown,  kept  on  ice. 

Holst§  observed  a  virulent  Streptococcus  brevis  that  remained 
unchanged  upon  artificial  culture-media  for  eight  years  without 
any  particular  precautions  having  been  taken  to  maintain  the 
virulence. 

Dried  streptococci  are  said  by  Frosch  and  Kolle||  to  retain  their 
virulence  longer  than  those  growing  on  culture-media. 

Metabolic  Products. — The  streptococcus  produces  a  ferment  by 
which  milk  is  coagulated.  A  few  streptococci  (S.  faecalis  of  And- 
rewes  and  Horder)  are  said  to  produce  gelatine  softening  ferments, 
but  this  Streptococcus  pyogenes  never  does. 

The  organisms  derive  O  from  the  atmosphere  or  from  compounds, 
but  no  gas  is  ever  evolved  in  the  process,  though  acids  are  always 
produced  in  the  presence  of  saccharose,  lactose,  rhamnose  (iso- 
dulcite)  rafnnose,  inulin,  amygdalin,  arbutin,  coniferin,  digitalin, 
helicin,  populin,  salicin,  glycerin,  sorbite  and  mannite  (Gordon). 

*  Amer.  Jour.  Med.  Sci.,  1910,  cxl,  516;  1912,  clxiv,  313;  Trans.  Asso.  Amer. 
Phys.,  1912,  xxvii,  157. 

t  "Ann.  de  1'Inst.  Pasteur,"  July  25,  1895,  p.  rx,  No.  7,  593. 

j  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  May  4, 1895,  Bd.xvm,No.  i6,p.55i. 

§  Ibid.," March  21,  1896,  Bd.  xrx,  No.  n. 

||  Fliigge's  "Die  Mikroorganismen." 


Streptococcus  Pyogenes  315 

No  acids  are  formed  from  starch,  glycogen,  arabin,  convolvulin, 
huperidin,  jalapin,  methyl  glucoside,  saponin,  glycol,  erythrite  or 
dulcite  (Gordon). 

Marmorek*  and  Lubenauf  found  that  cultures  of  the  strep- 
tococcus when  grown  in  bouillon  containing  glucose,  produced 
a  hemolytic  substance — streptokolysin — not  seemingly  present 
in  cultures  grown  in  ordinary  bouillon.  Besredkaf  found  that 
streptokolysin  was  produced  only  by  highly  virulent  cultures  of 
the  streptococcus  and  not  by  saprophytic  organisms  that  have  been 
for  some  time  under  cultivation  in  the  laboratory. 

Levin§  investigated  the  subject  thoroughly  and  found  that 
different  strains  of  streptococci  produced  streptokolysin  in  varying 
quantities,  that  its  production  is  entirely  independent  of  virulence, 
that  it  is  destroyed  by  heat  (37°C.  in  some  days;  55°C.  in  one-half 
hour) ;  that  acidity  of  the  nutrient  media  hinders  its  formation,  and 
that  it  is  intimately  associated  with  the  bodies  of  the  streptococci 
by  which  it  is  produced,  so  that  in  the  sediment  obtained  by  nitra- 
tion or  by  centrifugation  there  is  nearly  one  thousand  times  as 
much  as  in  the  filtered  fluid  culture.  The  streptokolysin  is  not 
destroyed  by  the  death  of  the  bacteria.  Antistreptokolysin  is  pres- 
ent in  antistreptococcus  serum. 

Toxic  Products. — The  toxic  products  of  the  streptococcus  are 
not  well  known.  Cultures  from  different  sources  vary  greatly  in 
the  effects  produced  by  hypodermic  or  intravenous  injection  after 
filtration  through  porcelain.  Killed  cultures  produce  a  much  more 
marked  effect  than  filtered  ones,  so  that  the  important  product 
must  be  an  endotoxin. 

Simon||  found  that  the  toxic  quality  of  the  bodies  of  strepto- 
cocci of  different  stocks  had  nothing  to  do  with  their  virulence. 
Simon**  also  found  that  the  toxic  products  of  the  streptococcus  were 
diverse  and  peculiar.  The  bodies  of  the  cocci  contained  an  intra- 
cellular  toxin  the  activity  of  which  was  independent  of  virulence. 
This  poison  is  liberated  only  when  the  bactericidal  activities  of  the 
body  act  upon  the  cocci.  The  cocci  also  excrete  a  toxic  substance 
whose  activity  is  greater  than  that  of  the  intracellular  toxin,  but 
whose  production  is  subject  to  great  variation  and  is  entirely  in- 
dependent of  the  intracellular  toxin.  The  toxins  and  hemolysins 
are  entirely  different  bodies. 

In  general,  the  effects  of  streptococcus  intoxication  are  vague. 
The  animals  appear  weak  and  ill,  and  have  a  slight  fever;  but  un- 
less the  virulence  of  the  culture  be  exceptional  or  the  dose  very  large, 
they  usually  recover  in  a  short  time. 

*  "Annales  de  PInst.  Pasteur,"  1895,  593. 
t  "Centralbl.  f.  Bakt.,"  etc.,  1901,  Bd.  xxx,  Nos.  9  and  10. 
j  "Ann.  de  1'Inst.  Pasteur,"  1901,  p.  880. 
§  "Nord.  Med.  Ark.,"  1903,  n,  No.  15,  p.  20. 
||  "Centralbl.  f.  Bakt.,"  Dec.  18,  1903,  xxxv,  No.  3,  p.  308. 
**  Ibid.,  Jan.  16,  1904,  xxxv,  No.  4,  p.  350. 


316  Suppuration 

Coley's  Mixture. — The  clinical  observation  that  occasional 
accidental  erysipelatous  infection  of  malignant  tumors  is  followed 
by  sloughing  and  the  subsequent  disappearance  of  the  tumor, 
suggested  the  experimental  inoculation  of  such  tumors  with  Strep- 
tococcus erysipelatis  as  a  therapeutic  measure.  The  danger  of  the 
remedy,  however,  caused  many  to  refrain  from  its  use,  for  when  one 
inoculates  the  living  erysipelas  virus  into  the  tissues  it  is  impossible 
to  estimate  the  exact  amount  of  disturbance  that  will  follow. 

To  overcome  this  difficulty  Coley*  has  recommended  that  the 
toxin  instead  of  the  living  coccus  be  used  for  injection. 

A  virulent  culture  of  the  streptococcus  is  obtained,  by  preference  from  a  fatal 
case  of  erysipelas,  inoculated  into  small  flasks  of  bouillon,  and  allowed  to  grow  for 
three  weeks.  The  flask  is  then  reinoculated  with  Bacillus  prodigiosus,  allowed 
to  grow  for  ten  or  twelve  days  at  the  room  temperature,  well  shaken  up,  poured 
into  bottle  of  about  f5ss  capacity,  and  rendered  perfectly  sterile  by  an  exposure 
to  a  temperature  of  50°  to  6o°C.  for  an  hour.  It  is  claimed  that  the  combined 
products  of  the  streptococcus  of  erysipelas  and  Bacillus  prodigiosus  are  much 
more  active  than  a  simple  streptococcus  culture.  The  best  effects  follow  the 
treatment  of  cases  of  inoperable  spindle-cell  sarcoma,  where  the  toxin  sometimes 
causes  a  rapid  necrosis  of  the  tumor  tissue,  which  can  be  scraped  out  with  an 
appropriate  instrument.  Numerous  cases  are  on  record  in  which  this  treatment 
had  been  most  efficacious;  but,  although  Coley  still  recommends  it  and  Czerny 
upholds  it,  the  majority  of  surgeons  have  failed  to  secure  the  desired  results. 

Antistreptococcus  Serum. — Since  1895  considerable  attention 
has  been  bestowed  upon  the  antistreptococcus  serum  of  Marmorekf 
and  Gromakowsky,!  which  is  said  to  act  specifically  upon  strepto- 
coccus infections,  both  general  and  local.  Numerous  cases  of 
suppuration,  septic  infection,  puerperal  fever,  and  scarlatina  are 
upon  record  in  which  the  serum  seems  to  have  exerted  a  beneficial 
action. 

The  serum  is  prepared  by  the  injection  of  cultures  of  living 
virulent  streptococci  into  horses,  until  a  high  degree  of  immunity  is 
attained.  The  serum  is  probably  both  antitoxic  and  bactericidal 
in  action. 

The  success  following  the  serums  of  some  experimenters  upon 
certain  cases,  and  their  occasional  or  constant  failure  in  other 
cases,  have  suggested  that  there  is  considerable  difference  between 
different  "strains"  or  families  of  streptococci.  To  obviate  this 
inequality  Van  de  Velde§  has  made  a  polyvalent  antistreptococcus 
serum  by  using  a  number  of  different  cultures  secured  from  the 
most  diverse  clinical  cases  of  streptococcus  infection.  Another 
serum,  of  Tavel||  and  Moser,**is  made  by  using  cultures  from  dif- 
ferent cases  of  scarlatina.  The  use  of  these  serums,  however,  has 
not  given  the  satisfaction  expected,  and  at  the  present  moment 
the  whole  subject  of  antistreptococcus  serums  is  debatable  both 

*  "  Amer.  Jour.  Med.  Sci.,"  July,  1894. 

t  "Ann.  de  Plnst.  Pasteur,"  July  25,  1895,  rx,  No.  7,  p.  593- 
Jlbid. 

§  "Archiv.  de.  med.  Exper.,"  1897. 
"-||  "Deutsche  med.  Wochenschrift,"  1903,  No.  50. 
**  "  Berliner  klin.  Wochenschrift,"  1902,  13. 


Streptococcus  Mucosus  317 

from  the  standpoint  of  its  theoretic  scientific  basis  and  its  thera- 
peutic application. 

Streptococcus  Vaccine. — Vaccines  made  by  the  method  given  in 
the  chapter  on  "  Bacterio- vaccines  "  are  now  used  in  all  streptococcus 
infections  with  varying  success.  As,  however,  there  is  no  knowl- 
edge by  which  one  can  foretell  exactly  what  course  a  streptococcus 
infection  will  pursue,  it  is  impossible  to  determine  with  accuracy 
what  advantage  results  from  the  treatment.  Judged  upon  its 
clinical  merits,  streptococcus  vaccine  does  good,  especially  when 
the  vaccine  is  homologous.  When  homologous  vaccine  cannot  be 
prepared,  preference  might  next  be  given  the  so-called  "polyvalent" 
vaccines  made  by  combining  cultures  from  many  sources.  Such, 
especially  when  "sensitized"  by  admixture  with  antistreptococcus 
serum,  according  to  the  method  of  Besredka,  give  promise  of  benefit 
upon  theoretical  grounds. 

STREPTOCOCCUS  Mucosus  (HOWARD  AND  PERKINS) 

This  organism,  described  by  Howard  and  Perkins,*  was  isolated 
from  a  case  of  tubo-ovarian  abscess  with  generalized  infection,  and 


Fig.  in. — Streptococcus  mucosus,  from  peritoneal  exudate.      X  1200 
(Howard  and  Perkins,  in  "Journal  of  Medical  Research"). 

again  later  by  Schottmiillert  from   a   case   of   parametritis,  peri- 
tonitis, meningitis,  and  phlebitis. 

It  occurs  as  a  rounded  coccus  in  pairs  and  in  short  chains,  though 
sometimes  long  chains  of  a  hundred  have  been  observed.  The  pairs 
resemble  gonococci.  They  measure  1.25  to  1.75  M  in  length  and 
0.5  to  0.75  fj,  in  breadth.  Each  is  surrounded  by  a  halo  that  varies 
in  width  from  1.5  to  3.0  ju,  which  shows  best  in  cultures  grown  on 

*  "Journal  of  Medical  Research,"  1901,  N.  S.  i,  163. 
t  "Munch,  med.  Wochenschrift,"  1903,  xxi. 


318  Suppuration 

human  blood-serum.  The  usual  capsule  stains  fail  to  color  this 
halo  when  the  organisms  are  from  artificial  cultures,  though  they 
show  it  well  when  they  are  in  pus.  The  organisms  stain  with  ordi- 
nary dyes  and  by  Gram's  method. 

The  cultures  resemble  those  of  Streptococcus  pyogenes,  but 
are  rather  more  luxuriant,  the  colonies  having  a  bluish  cast.  The 
organism  ferments  inulin,  which  makes  Hiss  think  it  related  to  the 
pneumococcus. 

The  organism  taken  at  autopsy  and  inoculated  into  the  peri- 
toneum of  a  guinea-pig  caused  the  animal  to  die,  comatose,  in 
thirty-six  hours,  with  peritonitis.  There  were  15  to  20  cc.  of 
peculiar  viscid  fluid  in  the  peritoneal  cavity.  It  had  a  grayish  puru- 
lent character  and  contained  numerous  flakes  of  fibrin.  There  was 
no  generalized  infection.  Mice  and  rabbits  were  susceptible  and 
died  of  generalized  infection. 

The  organism  is  not  infrequently  found  as  an  apparently  harm- 
less tenant  of  the  human  mouth,  where  it  may  be  confused  with 
the  pneumococcus.  It  has  also  turned  up  unexpectedly  in  a  variety 
of  inflammatory  diseases. 

STREPTOCOCCUS  ERYSIPELATIS  (FEHLEISEN) 

The  streptococcus  of  Rosenbach  is  generally  thought  to  be 
identical  with  a  streptococcus  described  by  Fehleisen*  as  Strepto- 
coccus erysipelatis. 

The  streptococcus  of  erysipelas  can  be  obtained  in  almost  pure 
culture  from  the  serum  which  oozes  from  a  puncture  made  in  the 
margin  of  an  erysipelatous  patch.  They  are  small  cocci,  usually 
forming  chains  of  from  six  to  ten  individuals,  but  sometimes  reach- 
ing a  hundred  or  more  in  number.  Occasionally  the  chains  occur 
in  tangled  masses. 

They  can  be  cultivated  at  the  room  temperature,  but  grow 
much  better  at  30°  to  37°C.  They  are  not  particularly  sensi- 
tive to  the  presence  or  absence  of  oxygen,  but  perhaps  develop 
a  little  more  rapidly  in  its  presence.  The  cultural  appearances  are 
identical  with  those  of  Streptococcus  pyogenes. 

When  injected  into  animals  Fehleisen's  coccus  behaves  exactly 
like  Streptococcus  pyogenes. 

MICROCOCCUS  TETRAGENUS  (GAFFKY) 

General  Characteristics. — Large,  round,  encapsulated  cocci,  regularly  asso- 
ciated in  groups  of  four,  forming  tetrads.  They  are  non-motile,  non-flagellated, 
non-sporogenous,  non-liquefying,  non-chromogenic,  non-aerogenic,  aerobic  and 
optionally  aerobic,  pathogenic  for  mice  and  other  small  animals,  and  stain  well 
by  all  methods,  including  that  of  Gram. 

A  large  micrococcus  grouped  in  fours    and   known  as   Micro- 
*  "Verhandlungen  der  Wurzburger  med.  Gesellschaft,"  1881. 


Streptococcus  Tetragenus 


coccus  tetragenus  can  sometimes  be  found  in  normal  saliva,  tuber- 
culous sputum,  and  more  commonly  in  the  contents  of  the  cavities 
of  tuberculosis  pulmonalis.  It  sometimes  occurs  in  the  pus  of 
acute  abscesses,  and  may  be  of  importance  in  connection  with  the 
pulmonary  abscesses  which  complicate  tuberculosis.  It  was  dis- 
covered  by  Gaffky.* 

Morphology. — The  cocci  are  rather  large,  measuring  about  i  jj, 
in  diameter.  In  cultures  they  do  not  show  the  regular  arrange- 
ment in  tetrads  as  constantly  as  in  the  blood  and  tissues  of  animals, 
where  they  occur  in  groups  of  four  surrounded  by  a  transparent 
gelatinous  capsule. 


Fig.  112. — Micrococcus  tetragenus  in  spleen  of  infected  mouse.     (From  Hiss 
and  Zinsser," Text-Book  of  Bacteriology,"  D.  Appleton  &  Co.,  Publishers.) 

Staining. — The  organisms  stain  well  by  ordinary  methods  and 
beautifully  by  Gram's  method,  by  which  they  can  best  be  demon- 
strated in  tissues. 

Isolation. — The  organism  can  be  isolated  by  inoculating  a  white 
mouse  with  sputum  or  pus  containing  it,  and  after  death  recovering 
it  from  the  blood. 

Cultivation. — It  grows  readily  upon  artificial  media.  Upon 
gelatin  plates  small  white  colonies  are  produced  in  from  twenty- 
four  to  forty-eight  hours.  Under  the  microscope  they  appear 

*  "Archiv.  f.  Chirurgie,"  xxvin,  3. 


320  Suppuration 

spheric  or  elongate  (lemon  shaped),  finely  granular,  and  lobulated 
like  a  raspberry  or  mulberry.  When  superficial  they  are  white  and 
elevated,  i  to  2  mm.  in  diameter. 

Gelatin. — In  gelatin  punctures  a  large  white  surface  growth 
takes  place,  but  development  in  the  puncture  is  very  scant,  the 
small  spheric  colonies  usually  remaining  isolated.  The  gelatin  is 
not  liquefied. 

Agar-agar. — Upon  agar-agar  spheric  white  colonies  are  produced. 
They  may  remain  discrete  or  become  confluent. 

Potato. — Upon  potato  a  luxuriant,  thick,  white  growth  is  formed. 

Blood-serum. — The  growth  upon  blood-serum  is  also  abundant, 
especially  at  the  temperature  of  the  incubator.  It  has  no  distinctive 
peculiarities. 


Fig.  113. — Micrococcus  tetragenus;  colony  twenty-four  hours  old  upon  the 
surface  of  an  agar-agar  plate.     X  100  (Heim). 

Pathogenesis. — The  introduction  of  tuberculous  sputum  or  of  a 
minute  quantity  of  a  pure  culture  of  this  coccus  into  white  mice 
usually  causes  a  fatal  bacteremia  in  which  these  organisms  are  found 
in  small  numbers  in  the  heart's  blood,  but  are  numerous  in  the 
spleen,  lungs,  liver,  and  kidneys. 

Japanese  mice  and  white  mice  are  highly  susceptible  to  the 
organism  and  die  three  or  four  days  after  inoculation. 

House-mice,  field-mice,  and  rabbits  are  comparatively  immune. 
Guinea-pigs  may  die  of  general  septic  infection,  though  local  ab- 
scesses result  from  subcutaneous  inoculation. 

The  tetracocci,  when  present,  probably  hasten  the  tissue-necrosis 
in  tuberculous  cavities,  aid  in  the  formation  of  abscesses  of  the  lung 
and  contribute  to  the  production  of  the  hectic  fever. 

An  interesting  contribution  to   the  relationship  of  this   coccus 
to  human  pathology  has  been  made  by  Lartigau,*  who  succeeded 
in  demonstrating  that  the  tetracoccus  may  be  the  cause  of  a  pseudo- 
membranous  aijgina,  3  cases  of  which  came  under  his  observation. 
*  "Phila.  Med.  Jour.,"  April  22,  1899. 


Bacillus  Pyocyaneus 


321 


Bezancon*  has  isolated  this  organism  from  a  case  of  meningitis. 
Forneacaj  has  reported  a  case  of  generalized  tetragenous  septicemia. 


BACILLUS  PYOCYANEUS   (GESSARD) 

General  Characteristics. — A  minute,  slender,  actively  motile,  flagellated,  non- 
sporogenous,  chromogenic  and  feebly  pathogenic,  aerobic  or  facultative  anaerobic, 
liquefying  bacillus,  staining  by  ordinary  methods,  but  not  by  Gram's  method. 

In  some  cases  pus  has  a  peculiar  bluish  or  greenish  color,  which 
depends  upon  the  presence  of  Bacillus  pyocyaneus  of  Gessard.J 

Distribution. — The  bacillus  appears  to  be  a  rather  common 
saprophyte,  being  found  in  feces,  manure,  and  water.  It  easily 


Fig.  114. — Bacillus  pyocyaneus,  from  an  agar-agar  culture.      X  1000 
(Itzerott  and  Niemann). 

takes  up  its  residence  upon  the  skin  and  mucous  membranes,  and 
has  been  found  in  the  perspiration.  It  sometimes  occurs  as  a  sapro- 
phyte upon  the  surgical  dressings  applied  to  wounds,  and  some- 
times invades  the  tissues  through  wounds,  to  occasion  dangerous 
infections. 

Morphology. — It  is  a  short,  slender  organism  with  rounded 
ends,  measuring  0.3  X  i  to  2  /x,  according  to  Flugge;  0.6  X  2  to 
6  IJL,  according  to  Ernst,  and  o..6  X  i  M>  according  to  Charriri.  It 
is  quite  pleomorphous,  which  probably  accounts  for  the  difference 
in  measurements.  It  is  occasionally  united  in  chains  of  four  or  six. 
It  is  actively  motile,  has  one  terminal  flagellum,  and  does  not  form 
spores. 

It  closely  resembles  a  harmless  bacillus  found  in   water,   and 

*  "Semaine  Medicale,"  1898. 

t  "Riforma  Medica,"  1903. 

t  "De  la  Pyocyanine  et  de  son  Microbe,"  These  de  Paris,  1882. 


322  Suppuration 

known  as  Bacillus  fluorescens  liquefaciens,  from  which  Ruzicka* 
thinks  it  has  probably  descended. 

Staining. — It  stains  well  with  the  ordinary  staining  solutions,  but 
not  by  Gram's  method. 

Isolation. — The  isolation  of  the  organism  is  simple,  the  ordinary 
plate  method  being  a  satisfactory  means  of  securing  it  from  pus 
or  other  discharges. 

Cultivation. — Colonies. — The  superficial  colonies  upon  gelatin 
plates  are  small,  irregular,  slightly  greenish,  ill-defined,  and  produce 
a  distinct  fluorescence  of  the  neighboring  medium. 

Microscopic  examination  shows  the  superficial  colonies  to  be 
rounded  and  coarsely  granular,  with  serrated  or  slightly  filamentous 
borders.  They  are  distinctly  green  in  the  center  and  pale  at  the 

'~ 


Fig.  115. — Bacillus  pyocyaneus.     Colonies- upon  gelatin  (Abbott). 

edges.  The  colonies  sink  into  the  gelatin  as  the  liquefaction  pro- 
gresses. Four  or  five  days  must  elapse  before  the  medium  is  all 
fluid. 

Gelatin  Punctures. — In  gelatin  puncture  cultures  the  chief  de- 
velopment of  the  organisms  occurs  at  the  upper  part  of  the  tube, 
where  a  deep  saucer-shaped  liquefaction  forms,  slowly  descending 
into  the  medium,  and  causing  a  beautiful  fluorescence.  At  times  a 
delicate  scum  forms  on  the  surface,  sinking  to  the  bottom  as  the 
culture  ages,  and  ultimately  forming  a  slimy  sediment. 

Agar-agar. — Upon  agar-agar  the  growth  developing  all  along 
the  line  of  inoculation  at  first  appears  bright  green.  The  green 
color  depends  upon  a  soluble  pigment  (fluorescin)  which  soon 
saturates  the  culture-medium  and  gives  it  the  characteristic  fluores- 
cent appearance.  As  the  culture  ages,  or  if  the  medium  upon  which 
it  grows  contains  much  peptone,  a  second  blue  pigment  (pyocyanin) 
develops,  and  the  bright  green  fades  to  a  deep  blue-green,  dark  blue, 
or  in  some  cases  to  a  deep  reddish-brown  color.  This  pigment  has 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  July  15,  1898,  p.  n. 


Bacillus  Pyocyaneus  323 

been  made  the  subject  of  a  careful  investigation  by  Jordan.*  Its 
formula,  according  to  Ledderhose,f  is  Ci4Hi4N2O. 

A  well-known  feature  of  the  growth  upon  fresh  agar-agar,  upon 
which  much  stress  has  recently  been  laid  by  Martin, {  is  the  forma- 
tion of  crystals  in  fresh  cultures.  Crystal  formation  in  cultures  of 
other  bacteria  usually  takes  place  in  old,  partially  dried  agar-agar, 
but  Bacillus  pyocyaneus  often  produces  crystals  in  a  few  days  upon 
fresh  media.  Freshly  isolated  bacilli  show  this  power  more  markedly 
than  those  which  have  been  for  some  time  part  of  the  laboratory 
stock  of  cultures  and  frequently  transplanted. 

Bouillon. — In  bouillon  the  organism  produces  a  diffuse  cloudi- 
ness, a  fluorescence,  and  sometimes  an  indefinite  thin  pellicle  on  the 
surface. 

Potato. — Upon  potato  a  luxuriant  greenish  or  brownish,  smeary 
layer  is  produced. 

Milk. — Milk  is  coagulated  and  peptonized.  It  is  slightly  acid 
for  the  first  day  or  two,  then  becomes  alkaline  again. 

Metabolic  Products. — Apart  from  the  pyocyanin  and  fluorescin, 
the  former  blue,  the  latter  green,  cultures  of  this  organism  frequently 
turn  red  brown.  This  suggested  the  formation  of  a  third  pigment, 
but  the  work  of  Boland§  has  shown  this  to  be  a  transformation  prod- 
uct of  pyocyanin  common  in  old  cultures. 

The  organism  produces  a  curdling  ferment,  a  fibrin-  and  casein- 
dissolving  ferment,  a  gelatin-dissolving  ferment,  and  a  bacteriolytic 
ferment,  the  pyocyanase  of  Emmerich  and  Low. 

It  also  produces,  under  favorable  conditions,  a  toxin  which  has 
been  studied  by  Wassermann,  who  found  it  fatal  in  doses  of  0.2 
to  0.5  cc.  when  intraperitoneally  injected  into  guinea-pigs.  The 
animals  show  peritonitis  and  punctiform  hemorrhages  on  the  serous 
membranes. 

Bullock  and  Hunter||  found  that  Bacillus  pyocyaneus  also  pro- 
duces a  hemolytic  substance,  pyocyanolysin,  by  which  corpuscles  of 
man,  oxen,  sheep,  apes,  rabbits,  cats,  rats,  dogs,  and  mice  are  dis- 
solved. The  peculiar  substance  was  produced  in  greatest  quantity 
in  virulent  cultures  three  or  four  weeks  old.  Jordan**  believes  that 
this  hemolytic  property  depends  solely  upon  the  intense  alkali 
formed  in  old  cultures.  Gheorghewskiff  found  a  leukocyte-destroy- 
ing substance  in  the  cultures. 

In  addition  to  the  metabolic  pigments  mentioned,  the  organism 
produces  toxins.  Wassermann  tt  found  that  filtrates  of  old  cultures 
were  more  toxic  for  guinea-pigs  than  the  endotoxins  made  by  lysis 

*  "Journal  of  Experimental  Medicine,"  1899,  vol.  rv. 

t  "Deutsche  Zeitschr.  f.  Chirurgie,"  1888,  Bd.  xxvm. 

j  "Centralbl.  f.  Bakt.,"  April  6,  1897,  xxi,  p.  473. 

§  "Centralbl.  f.  Bakt.,"  1899,  Bd.  xxv,  p.  897. 

||  Ibid.,  xxvm,  1900,  p.  865. 
**  Ibid.,  Bd.  xxxm,  Ref.  1903. 
ft  "Ann.  de  1'Inst.  Pasteur,"  1899,  xm. 
Jt  "Zeitschrift  fur  Hygiene,  1896,  xxn. 


324  Suppuration 

of  dead  bacteria.  The  organism  thus  produces  both  endo-  and 
exotoxins. 

Pathogenesis. — The  bacillus  is  pathogenic  for  the  small  laboratory 
animals,  but  different  cultures  differ  greatly  in  virulence.  One 
cc.  of  a  virulent  bouillon  culture,  injected  into  the  subcutaneous 
tissue  of  a  guinea-pig,  causes  rapid  edema,  suppurative  inflamma- 
tion, and  death  in  a  short  time  (twenty-four  hours).  Sometimes  the 
animal  lives  for  a  week  or  more,  then  dies.  There  is  a  marked 
hemorrhagic  subcutaneous  edema  at  the  seat  of  inoculation.  The 
.bacilli  can  be  found  in  the  blood  and  in  most  of  the  tissues. 
Rats  and  mice  behave  similarly  to  guinea-pigs  when  inoculated 
subcutaneously. 

Rabbits  are  less  susceptible  and  subcutaneous  injections  rarely 
cause  death.  Intraperitoneal  injection  may  be  followed  by  fatal 
infection  if  the  bacillus  be  highly  virulent  or  if  it  be  not  virulent, 
recovery  may  occur.  Intravenous  inoculation  causes  fever,  al- 
buminuria,  diarrhea  and  death  in  a  day  or  two.  If  the  dose  be 
smaller  or  the  virulence  of  the  culture  less,  a  subacute  disturbance 
characterized  by  wasting,  palsy  and  convulsions  may  occur.  If 
the  animal  dies,  nephritis  can  usually  be  found,  and  perhaps  explains 
the  symptoms. 

Dogs  are  susceptible  to  infection  by  B.  pyocyaneus,  the  symptoms 
bearing  a  considerable  resemblance  to  rabies. 

Blum*  reports  a  case  of  pyocyaneus  infection  with  endocarditis 
in  a  child. 

Lartigau,t  in  his  study  of  "The  Bacillus  Pyocyaneus  as  a  Factor 
in  Human  Pathology,"  sums  up  what  is  known  about  this  role  of 
the  organism  as  follows: 

"The  Bacillus  pyocyaneus,  like  many  pathogenic  micro-organisms,  is  occasion- 
ally found  in  a  purely  saprophytic  role  in  various  situations  in  the  human 
economy.  It  has  been  found  in  the  saliva  by  Pansini,  in  sputum  by  Frisch, 
and  in  the  sweat  by  Eberth  and  Audanard.  Abelous  demonstrated  its  pres- 
ence in  the  stomach  as  a  saprophyte.  Its  existence  in  suppurating  wounds 
has  long  been  known,  and  Koch  early  detected  its  presence  in  tuberculous 
cavities,  regarding  it  as  an  organism  incapable  of  playing  any  pathologic  role. 
The  etiologic  relation  of  the  organism  to  certain  cases  of  purulent  otiti? 
media  in  children  was  pointed  out  by  Martha,  Maggiora  and  Gradenigo, 
Babes,  Kossel,  and  others.  H.  C.  Ernst  obtained  it  from  a  pericardial  exudate 
during  life.  G.  Blumer  demonstrated  its  presence  in  practically  pure  cultures 
in  a  case  of  acute  angina  simulating  diphtheria;  Jadkewitsch,  B.  Motz,  and  Le 
Noir  obtained  the  bacillus  in  cases  of  urinary  infection.  The  cases  of  Triboulet, 
Karlinski,  Oettinger,  Ehlers,  and  Barker  are  interesting  instances  of  its  role  in 
cutaneous  lesions. 

"In  addition  to  these  lesions,  other  morbid  processes  have  been  associated  in 
some  cases  with  the  bacillus  of  blue  pus,  such  as  meningitis  and  bronchopneu- 
monia,  by  Monnier;  diarrhea  of  infants,  by  Neumann,  Williams,  Thiercelin  and 
Lesage,  and  other  observers;  dysentery,  by  Calmette  and  by  Lartigau;  and 
general  infection,  by  Ehlers,  Neumann,  Oettinger,  Karlinski,  Monnier,  Krannhals, 
Calmette.  Finkelstein,  and  L.  E.  Barker." 

Nine  additional  cases  of  human  infection  are  reported  by  Perkins.}; 

*  "-Centralbi.  f.  Bakt.  u.  Parasitenk.,"  Feb.  10,  1899,  xxv,  No.  4. 

t  "Phila.  Med.  Jour.,"  Sept.  17,  1898. 

j  "Jour,  of  Med.  Research,"  1901,  vol.  vi,  p.  281. 


Bacillus  Proteus  Vulgaris  325 

Immunity. — Immunity  against  pyocyaneus  infection  develops 
after  a  few  inoculations  with  attenuated  or  sterilized  cultures. 
These  are  easily  prepared,  the  thermal  death-point  determined  by 
Sternberg  being  56°C.  It  also  follows  injection  of  either  the  endo- 
toxin  or  the  exotoxin.  In  the  immunity  resulting  from  the  treat- 
ment with  bacterio-vaccines  the  serum  of  the  animal  becomes 
agglutinative  and  bactericidal;  in  the  immunity  resulting  from 
treatment  with  the  exotoxin,  antitoxin  is  produced. 

BACILLUS  PROTEUS  VULGARIS  (HAUSER) 

General  Characteristics. — An  actively  motile,  flagellated,  non-sporogenous, 
non-chromogenic,  liquefying,  aerobic  and  optionally  anaerobic,  doubtfully 
pathogenic,  aerogenic  bacillus,  easily  cultivated  on  artificial  media  and  readily 
stained  by  the  ordinary  methods,  though  not  by  Gram's  method. 

This  bacillus  was  first  found  by  Hauser*  in  decomposing  animal 
infusions,  usually  in  company  with  two  closely  allied  forms,  Proteus 
mirabilis  and  Proteus  zenkeri,  which,  as  the  experiments  and 
observations  of  Sanfelice  and  others  show,  may  be  identical  with  it. 
According  to  Kruse,  it  is  quite  probable  that  the  mixed  species 
formerly  called  Bacterium  termo  was  largely  made  up  of  the  proteus. 

Distribution. — The  organism  is  a  common  saprophyte  and  is 
very  abundant  in  water,  earth,  and  air.  It  is  to  be  expected  wher- 
ever putrefactive  change  is  in  progress.  It  is  a  common  mistake  for 
the  novice  to  look  upon  it  as  a  member  of  the  Bacillus  coli  group. 

Morphology. — The  bacilli  are  variable  in  size  and  shape — pleo- 
morphic — and  are  named  proteus  from  this  peculiarity.  Some  differ 
very  little  from  cocci,  some  are  more  like  the  colon  bacillus  in  shape, 
others  form  long  filaments,  and  occasional  spirulina  forms  are  met 
with.  True  spirals  are  never  found.  All  of  the  forms  mentioned 
may  be  found  in  pure  cultures  of  the  same  organism.  The  diameter 
of  the  bacillus  is  usually  about  0.6  ju,  but  the  length  varies  from  1.2 
/x  or  less  to  4  ju  or  more.  No  spores  are  formed.  The  organisms  are 
actively  motile.  The  long  filaments  frequently  form  loops  and 
tangles.  Flagella  are  present  in  large  numbers.  Upon  one  of  the 
long  bacilli  as  many  as  one  hundred  have  been  counted.  Involution 
forms  are  frequent  in  old  cultures. 

Staining. — The  bacilli  stain  well  by  the  ordinary  methods. 
Gram's  method  usually  fails. 

Cultivation. — The  proteus  is  easily  cultivated  and  grows  well  in 
all  the  artificial  media. 

Colonies. — Upon  gelatin  plates  a  typical  phenomenon  is  observed 
in  connection  with  the  development  of  the  colonies,  for  the  most 
advantageous  observation  of  which  the  medium  used  for  making 
the  cultures  should  contain  5  instead  of  10  per  cent,  of  gelatin. 
Kruse f  describes  the  phenomenon  as  follows: 

*  "Ueber  Faulnissbakterien,"  Leipzig,  1885. 
t  Flugge's  "Die  Mikroorganisrnen." 


326  Suppuration 

"At  the  temperature  of  the  room,  rounded,  saucer-shaped  depressions,  with 
a  whitish  central  mass  surrounded  by  a  lighter  zone,  are  quickly  formed.  Under 
low  magnification  the  center  of  each  is  seen  to  be  surrounded  by  radiations  ex- 
tending in  all  directions  into  the  solid  gelatin,  and  made  up  of  chains  of  bacilli. 
Between  the  radiations  and  the  granular  center  bacteria  are  seen  in  active  motion. 
Upon  the  surface  the  colony  extends  as  a  thin  patch,  consisting  of  a  layer  of 
bacilli  arranged  in  threads,  sending  numerous  projections  from  the  periphery. 
Under  certain  conditions  the  wandering  of  the  processes  can  be  directly  observed 
under  the  microscope.  It  depends  not  only  upon  the  culture-medium,  but,  in 
part,  upon  the  culture  itself.  Entire  groups  of  bacilli  or  single  threads,  by 
gradual  extension  and  circular  movement,  detach  themselves  from  the  colony  and 
wander  about  upon  the  plate.  From  the  radiated  central  part  of  the  colony 
peculiar  zooglea  are  formed,  having  a  sausage  or  screw  shape,  or  wound  in  spirals 
like  a  corkscrew.  The  younger  colonies,  which  have  not  yet  reached  the  surface 
of  the  gelatin,  are  more  compact,  rounded  or  nodular,  later  covered  with  hair-like 
projections,  and  becoming  radiated  like  the  superficial  colonies." 


Fig.  116. — Swarming  islands  of  proteus  bacilli  on  the  surface  of  gelatin;  X  650 

(Hauser). 

If  the  culture-medium  be  concentrated,  or  the  culture  have  been 
frequently  transplanted,  the  phenomenon  is  less  marked  or  may 
not  occur. 

Bouillon. — In  this  medium  the  organism  grows  rapidly,  and 
quickly  clouds  the  fluid.  A  pellicle  soon  forms  upon  the  surface  and 
a  mucilaginous  sediment  occurs  later. 

Gelatin  Punctures. — Puncture  cultures  in  gelatin  are  not  char- 
acteristic. A  stocking-like  liquefaction  occurs  in  the  gelatin  and 
extends  so  rapidly  that  the  entire  medium  is  liquefied  in  a  few  days. 
Anaerobic  cultures  do  not  liquefy. 

Agar-agar. — Upon  agar-agar  the  bacillus  forms  a  moist,  thin, 
transparent,  rapidly  extending  layer  which  rarely  reaches  the  sides 
of  the  tube.  Upon  agar-agar  plates  ameboid  movement  of  the 
colonies  sometimes  occurs. 


Amebae  and  Suppuration  327 

Potato.  —  Upon  potato  the  growth  occurs  in  the  form  of  a  smeary 
patch  of  soiled  appearance. 

Milk  is  coagulated. 

Metabolic  Products.  —  The  bacillus  usually  produces  alkalies. 
Indol  and  phenol  are  formed  from  the  peptone  of  the  culture- 
media.  Nitrates  are  reduced  to  nitrites,  and  then  partly  reduced 
to  ammonia.  In  most  culture-media  not  containing  sugar  the 
bacillus  produces  a  disagreeable  odor, 

In  culture-media  containing  either  grape-  or  cane-sugar  fermenta- 
tion occurs  both  in  the  presence  and  in  the  absence  of  oxygen. 
Milk-sugar  is  not  decomposed. 

Pathogenesis.  —  It  is  a  question  whether  or  not  Bacillus  proteus 
is  to  be  ranked  among  the  pathogenic  bacteria.  Small  doses  are 
harmless  for  the  laboratory  animals;  large  doses  produce  abscesses. 
A  toxic  substance  resulting  from  the  metabolism  of  the  organism 
seems  to  be  the  cause  of  death  when  considerable  quantities  of  a  cul- 
ture are  injected  into  the  peritoneal  cavity  or  blood-vessels.  The 
bacilli  do  not  seem  able  to  multiply  in  the  healthy  animal  body,  but 
can  do  so  when  previous  disease  or  injury  of  its  tissues  has  taken 
place. 

The  proteus  has  been  secured  in  cultures  from  wound  and  puerperal 
infections,  purulent  peritonitis,  endometritis,  and  pleurisy.  When 
the  local  lesion  is  limited,  as  in  endometritis,  the  danger  of  toxemia 
is  slight;  but  when  widespread,  as  the  peritoneum,  it  may  prove 
serious.  Bacillus  proteus  has  also  been  found  in  acute  infectious 
jaundice  and  in  acute  febrile  icterus,  or  Weil's  disease. 

Bordoni-Uffredizzi  has  shown  that  the  proteus  quite  regularly 
invades  the  tissues  after  death,  though  it  appears  unable  to  main- 
tain an  independent  existence  in  the  tissues  during  life,  and  is 
probably  of  importance  only  when  present  in  association  with  other 
bacteria.  It  at  times  grows  abundantly  in  the  urine,  and  may  pro- 
duce primary  inflammation  of  the  bladder.  The  inflammatory 
process  may  also  extend  from  the  bladder  to  the  kidney,  and  so 
prove  quite  serious. 

Epidemics  of  meat-poisoning  have  been  thought  to  depend  upon 
Bacillus  proteus.  One  of  them  was  studied  by  Wesenberg,*  who 
cultivated  the  organism  from  the  putrid  meat  by  which  63  persons 
were  made  ill.  Silverschmidtf  and  PfuhlJ  have  made  similar  in- 
vestigations with  similar  results. 


AND  SUPPURATION 

The  process  of  suppuration  is  not  confined  to  bacterial  micro- 
organisms, but  is  shared  to  a  limited  extent  by  the  protozoa.     Thus, 

*  "Zeitschrift  fur  Hygiene,"  etc.,  1898,  xxvm. 
t  Ibid.,  1899,  xxx. 
t  Ibid.,  1900,  xxxv. 


328  Suppuration 

Entamoeba  histolytica  (q.v.)  is,  to  all  appearances,  the  sole  excitant 
of  the  abscesses  of  the  liver  secondary  to  dysentery.  It  is  true  that 
these  are  cold  abscesses  and  necrotic  rather  than  distinctly  purulent 
in  character,  yet  it  seems  best  to  speak  of  the  organism  in  this 
connection. 

Entamoeba  buccalis  (Prowazek*)  is  a  small  ameba  that  has  been 
found  in  purulent  exudates  in  the  oral  tissues  of  persons  with  carious 
teeth.  It  is  at  present  thought  to  be  the  cause  of  Riggs'  disease  or 
pyorrhea  alveolaris. 

Amoeba  kartulisi  (Doneinf)  appears  to  be  capable  of  exciting 
suppuration.  It  was  found  by  Kartulis  in  the  pus  from  an  abscess 
of  the  right  side  of  the  lower  jaw.  The  patient  was  a  man  aged 
forty-three  years  who  had  been  operated  upon  for  the  removal  of  a 
piece  of  bone.  It  is  30  to  38  M  in  diameter,  is  actively  motile.  Its 
coarse  protoplasm  contains  red  and  white  blood-corpuscles.  Kar- 
tulis J  found  the  same  organism  five  times  in  other  cases,  and 
Flexner§  found  it  also. 

Amoeba  mortinatalium,  described  by  Smith  and  Weidman,|| 
was  found  in  distributed  small  purulent  foci  in  the  kidneys  and 
other  organs  of  a  still-born  fetus. 

MISCELLANEOUS   ORGANISMS  OF  SUPPURATION  DESCRIBED   MORE 
FULLY  ELSEWHERE 

Before  leaving  the  subject,  attention  must  be  directed  to  other 
bacteria  that  under  exceptional  circumstances  become  the  cause  of 
suppuration.  Among  these  are  the  pneumococcus  of  Frankel  and 
Weichselbaum,  the  typhoid  bacillus,  and  the  Bacillus  coli  communis. 
These  organisms  are  considered  under  separate  and  appropriate 
headings,  to  which  the  reader  is  advised  to  refer. 

*  "  Arbeiten  a.  d.  Kaiserl.  Gesundh.  Amt.,"  1904,  xxi,  i,  Bull.  p.  42. 
t  "Die  Protozoa  als  Krankheitserreger,"  Jena.,  1901,  p.  30. 
j  "Centralbl.  f.  Bakt.  u.  Parasitenk."  1903,  xxxni,  p.  471. 
~  "Bulletin  of  the  Johns  Hopkins  Hospital,"  1892,  xxv. 
"University  of  Pennsylvania  Medical  Bulletin,"  Sept.,  1910. 


CHAPTER  II 
MALIGNANT  EDEMA 

BACILLUS  (EDEMATIS  MALIGNI  (KOCH) 

General  Characteristics. — A  motile,  flagellated,  sporogenous,  anaerobic, 
liquefying,  aerogenic,  non-chromogenic,  pathogenic  bacillus  of  the  soil,  readily 
stained  by  the  ordinary  methods,  but  not  by  Gram's  method. 

This  organism  was  originally  found  by  Pasteur*  in  putrescent 
animal  infusions  and  called  by  him  (1875)  Vibrion  septique.  It  was 
later  more  carefully  studied  and  described  by  Koch.f 

It  is  supposed  that  this  bacillus  was  among  the  organisms  whose 
introduction  into  wounds  in  the  days  of  pre-antiseptic  surgery, 
commonly  occasioned  the  then  prevalent  " Hospital  gangrene." 

Distribution. — The  organism  is  widely  distributed  in  nature, 
being  commonly  present  in  garden  earth.  It  is  also  found  in  dust, 
in  waste  water  from  houses,  and  sometimes  in  the  intestinal  contents 
of  animals. 

Morphology. — The  bacillus  of  malignant  edema  is  a  large  rod- 
shaped  organism  with  rounded  ends,  measuring  2  to  10  JJL  by  0.8 
to  i.o  At.  It  is  usually  motile,  and  possesses  many  flagella.  It  pro- 
duces oval  endospores  centrally  situated  and  giving  a  barrel  shape 
to  the  parent  bacillus. 

Staining. — The  bacillus  stains  well  with  ordinary  cold  aqueous 
solutions  of  the  anilin  dyes,  but  not  by  Gram's  method. 

Cultivation. — The  organism  is  a  strict  anaerobe,  but  under  con- 
ditions by  which  provision  is  made  for  the  removal  of  oxygen, 
grows  well  both  at  the  room  temperature  and  at  that  of  the  incu- 
bator. It  is  not  difficult  to  secure  in  pure  culture,  being  most  easily 
obtained  from  the  edematous  tissues  of  guinea-pigs  and  rabbits 
inoculated  with  garden  earth. 

Colonies. — The  colonies  which  develop  upon  the  surface  of 
gelatin  kept  under  anaerobic  conditions  appear  to  the  naked  eye 
as  small  shining  bodies  with  liquid,  grayish-white  contents.  Under 
the  microscope  they  appear  filled  with  a  tangled  mass  of  long  fila- 
ments which  under  a  high  power  exhibit  active  movement.  The 
edges  of  the  colony  have  a  fringed  appearance,  much  like  the  colonies 
of  the  hay  or  potato  bacillus. 

Gelatin. — In  gelatin  tube  cultures  the  characteristic  growth 
cannot  be  observed  unless  the  tube  be  placed  under  anaerobic 

*  "Bull.  Acad.  Med.,"  1877  and  1881. 

t  "Mittheilungen  aus  dem  kaiserl.  Gesundheitsamte,"  I,  53. 

329 


33° 


Malignant  Edema 


conditions.  The  best  preparation,  therefore,  is  made  by  heating 
the  gelatin  to  expel  any  air  it  may  contain,  inoculating  it  while  still 
liquid,  and  solidifying  it  in  cold  (iced)  water.  In  such  a  tube  the 
bacilli  develop  in  globular  circumscribed  areas  of  cloudy  liquefaction 
which  contain  a  small  amount  of  gas.  In  gelatin  to  which  a  little 
grape-sugar  has  been  added  the  gas  production  is  marked. 

Agar-agar. — The  growth  takes  place  in  the  form  of  a  cloudy 
stream,  in  the  lower  part  of  deep  punctures  in  recently  heated 
agar-agar,  from  which  the  air  has  been  expelled.  If  the  agar-agar 
contains  i  per  cent,  of  glucose,  it  is  soon  split  up  by  the  gas  for- 
mation. Such  cultures  give  off  a  very  disagreeable  cdor. 


Fig.  117. — Bacillus  of  malignant  edema,  from  the  body-juice  of  a  guinea-pig 
inoculated  with  garden  earth.      X  1000  (Frankel  and  Pfeiffer). 

Bouillon. — In  deep  tubes  of  recently  heated  bouillon  a  diffuse 
turbidity  occurs  in  about  twenty-four  hours.  After  the  third  day 
the  upper  half  clears,  the  bacilli  and  spores  sedimenting  or  moving 
away  from  the  oxygen.  The  culture  gives  off  a  very  disagreeable 
odor. 

In  glucose  or  other  sugar  bouillon  in  the  fermentation  tube, 
considerable  gas  is  formed. 

The  gas  is  partly  inflammable,  partly  not. 

Milk. — Milk  is  slowly  coagulated. 

Potato. — The  bacillus  grows  upon  the  surface  of  potato  if  kept 
under  anaerobic  conditions. 

Blood-serum. — Upon  coagulated  blood-serum,  and  upon  coagu- 
lated egg-white,  growth  occurs  under  anaerobic  conditions,  both 
media  being  slowly  digested  and  softened. 

Vital  .Resistance. — The  bacilli  themselves  soon  succumb  when  ex- 
posed to  the  air.  They  are  destroyed  in  a  few  moments  by  heating 


Lesions  331 

to  6o°C.  The  spores,  on  the  other  hand,  resist  drying  and  exposure 
to  the  atmosphere  well  and  can  be  kept  alive  for  years  in  garden 
earth.  The  complete  destruction  of  the  spores  requires  exposure  to 
90°C.  for  a  half  hour.  Moist  heat  at  ioo°C.  kills  them  in  a  few 
minutes. 

Metabolic  Products. — Of  the  toxic  products  of  the  organism 
nothing  definite  is  known.  It  decomposes  albumin,  forming  fatty 
acids,  leucin,  hydroparacumaric  acid,  and  an  oil  with  an  offensive 
odor.  Among  the  gases  formed,  carbonic  acid,  hydrogen,  and  marsh 
gas  have  been  detected. 

Pathogenes;s. — When  introduced  beneath  the  skin,  the  bacillus 
is  pathoger  T  a  large  number  of  animals — mice,  guinea-pigs, 
rabbits,  h  dogs,  sheep,  goats,  pigs,  calves,  chickens,  and 

pigeons.  ^  e  seem  to  be  immune. 

Giinther  points  out  that  the  simple  inoculation  of  the  bacillus  upon 
an  abraded  surface  is  insufficient  to  produce  infection,  because  the 


Fig.  1 18.— Bacillus  cedematis,  dextrose  gelatin  culture  (Gunther). 

presence  of  oxygen  is  detrimental  to  its  growth.  When  the  bacilli 
are  deeply  introduced  beneath  the  skin,  infection  occurs. 

Mice,  guinea-pigs,  and  rabbits  sicken  and  die  in  about  forty-eight 
hours. 

Washed  spores  of  the  bacillus  are  quickly  taken  up  by  phagocytes 
and  destroyed  without  producing  infection.  Salt-solution  suspen- 
sions of  such  spores  quickly  infect,  however,  if  mixed  with  some 
tissue-injuring  agent  such  as  lactic  acid,  or  if  combined  with  a 
harmless  micro-organism  such  as  Bacillus  prodigiosus  by  which  the 
phagocytic  activity  of  the  leukocytes  is  distracted  through  preference. 

Lesions. — In  the  blood  the  bacilli  are  few  because  of  the  loosely 
combined  oxygen  it  contains.  The  great  majority  of  the  bacilli 
occupy  the  subcutaneous  tissue,  where  very  little  oxygen  is  present 
and  the  conditions  of  growth  are  good.  The  autopsy  shows  a 
marked  subcutaneous  edema  containing  immense  numbers  of  the 


332  Gaseous  Edema 

bacilli.  If  the  animal  be  permitted  to  remain  undisturbed  for  some 
time  after  death,  the  bacilli  spread  to  the  circulatory  system  and  reach 
all  the  organs. 

Brieger  and  Ehrlich*  have  reported  2  cases  of  malignant  edema 
in  man.  Both  occurred  in  typhoid  fever  patients  subcutaneously 
injected  with  musk,  the  infection  no  doubt  resulting  from  impurities 
in  the  therapeutic  agent. 

Grigorjeff  and  Ukkej  have  observed  another  interesting  case  of 
typhoid  fever  with  intestinal  ulcerations,  through  which  infection 
by  the  bacillus  of  malignant  edema  took  place.  The  case  was 
characterized  by  interstitial  emphysema  of  the  subcutaneous  tissue 
of  the  neck  and  breast,  gas  bubbles  in  the  muscles,  and  a  transforma- 
tion of  the  entire  liver  into  a  spongy  porous  mass  of  a  grayish-brown 
color.  The  spleen  was  enlarged  and  soft,  and  contained  a  few  gas- 
bubbles.  Though  the  writers  consider  this  organism  to  be  the  bacillus 
of  malignant  edema,  the  general  impression  one  receives  from  the 
description  of  the  lesions  suggests  that  it  was  Welch's  Bacillus 
aerogenes  capsulatus. 

Immunity. — Cornevin  found  that  the  passage  of  the  bacillus 
through  white  rats  diminished  its  virulence,  and  that  the  animals 
of  various  species  that  recovered  were  immune  against  subsequent 
infection  with  the  virulent  organisms.  Roux  and  Chamberlandt 
found  that  the  filtered  cultures  were  toxic  and  that  animals  could  be 
immunized  by  injection  with  this  toxic  filtrate. 

GASEOUS  EDEMA 

BACILLUS  AEROGENES  CAPSULATUS  (WELCH) 

General  Characteristics. — A  large,  stout,  non-motile,  non-flagellate,  sporogen- 
ous,  non-chromogenic,  purely  anaerobic,  markedly  aerogenic,  doubtfully  patho- 
genic bacillus,  easily  cultivated  in  artificial  media,  readily  stained  by  the  ordinary 
methods  and  by  Gram's  method. 

This  disease  is  caused  by  an  interesting  micro-organism  described 
by  Welch,  and  subsequently  studied  by  Welch  and  Nuttall,§  Welch 
and  Flexner,||and  others.  Welch  said  at  the  meeting  of  the  Society 
of  American  Bacteriologists  held  at  Philadelphia,  December  30,  1904, 
that  he  believed  this  organism  to  be  identical  with  Kline's  Bacillus 
enteritidis  sporogenes,**and  that  it  belongs  to  the  butyric  acid  group. 
It  is  probably  also  identical  with  Bacillus  phlegmone  emphysematose 
of  Frankel.ft  In  many  systematic  writings  the  organism  is  now 
called  Bacillus  welchii.  English  writers  identify  it  with  Bacillus 

*"  Berliner  klin.  Wochenschrift,"  1882,  No.  44- 
f  " Militar-medizin.  Jour.,"  1898,  p.  323. 
j  "Ann.  de  1'Inst.  Pasteur,"  1887. 

§  Bull,  of  the  Johns  Hopkins  Hospital,"  July  and  Aug.,  1892,  vol.  vm,  No.  24. 
||  "Jour,  of  Experimental  Medicine,"  Jan.,  1896,  vol.  I,  No.  i,  p.  6. 
**  Ceirtralbl.  f.'  Bakt.  u.  Parasitenk,  1895,  xvm,  737. 
-  ft  "Centralbl.  f.  Bakt.,"  etc.,  Bd.  xm,  p.  13. 


Morphology  333 

perfringens  of  Veillon  and  Zuber,*  and  Besson  describes  it  under 
this  name.  Pending  final  decision  upon  the  identity  of  these  organ- 
isms, it  is  here  called  by  the  name  originally  given  it  by  Welch 
who  first  secured  it  from  the  body  of  a  man  dying  suddenly  of 
aortic  aneurysm  with  a  peculiar  gaseous  emphysema  of  the  sub- 
cutaneous tissues  and  internal  organs,  and  a  copious  formation  of 
gas  in  the  blood-vessels.  The  blood  was  thin  and  watery,  of  a  lac 
color,  and  contained  many  large  and  small  gas  bubbles,  and  many 
bacilli,  which  were  also  obtained  from  it  and  the  various  organs, 
especially  in  the  neighborhood  of  the  gas  bubbles,  in  nearly  pure 
culture.  The  coloring-matter  of  the  blood  was  dissolved  out  of  the 
corpuscles  and  stained  the  tissues  a  deep  red. 

Distribution. — It  is  believed  that  the  natural  habitat  of  the  bacillus 
is  the  soil,  but  there  is  reason  to  think  that  it  commonly  occurs  in  the 
intestine,  and  may  occasionally  be  found  upon  the  skin. 


Fig.  119. — Bacillus  aerogenes  capsulatus  (from  photograph  by 
Prof.  Simon  Flexner). 

Morphology. — The  bacillus  is  a  large  organism,  measuring  3-5  n 
in  length,  about  the  thickness  of  the  anthrax  bacillus,  with  ends 
slightly  rounded,  or,  when  joined,  square.  It  occurs  chiefly  in  pairs 
and  in  irregular  groups,  but  may  also  occur  in  chains.  In  culture 
media  it  is  usually  straight,  with  slightly  rounded  ends.  In  old 
cultures  the  rods  may  be  slightly  bent,  and  involution  forms  occur. 
The  bacillus  varies  somewhat  in  size,  especially  in  length,  in  different 
culture-media.  It  usually  appears  thicker  and  more  variable  in 
length  in  artificial  cultures  than  in  the  blood  of  animals. 

The  bacillus  is  not  motile  and  has  no  flagella. 

Dunham  f  found  that  spores  were  produced  upon  blood-serum,  and 
especially  upon  Loffler's  blood-serum  bouillon  mixture.  The  spores 

*  Archiv  de  med.  exper.  et  d'anat.  path.,  1898,  x,  517. 

f  "Bull,  of  the  Johns  Hopkins  Hospital,"  April,  1897,  p.  68. 


334  Gaseous  Edema 

resist  desiccation  and  exposure  to  the  air  for  ten  months.  They 
stain  readily  in  hot  solutions  of  fuchsin  in  anilin  water,  and  are  not 
decolorized  by  a  moderate  exposure  to  the  action  of  3  per  cent,  solu- 
tion of  hydrochloric  acid  in  absolute  alcohol.  They  are  oval,  and 
are  usually  situated  near  the  middle  of  the  bacillus,  which  is  distended 
because  of  the  large  size  of  the  spore  and  bulges  at  the  sides. 

Staining. — The  organism  stains  well  with  the  ordinary  stains, 
and  retains  the  color  well  in  Gram's  method.  When  stained  with 
methylene-blue  a  granular  or  vacuolated  appearance  is  sometimes 
observed,  due  to  the  presence  of  unstained  dots  in  the  cytoplasm. 

Usually  in  the  body-fluids  and  often  in  cultures  the  bacilli  are 
surrounded  by  distinct  capsules — clear,  unstained  zones.  To  dem- 
onstrate this  capsule  to  the  best  advantage,  Welch  and  Nuttall  de- 
vised the  following  special  stain: 

A  cover  is  thinly  spread  with  the  bacilli,  dried,  and  fixed  without 
overheating.  Upon  the  surface  prepared,  glacial  acetic  acid  is 
dropped  for  a  few  moments,  then  allowed  to  drain  off,  and  at  once 
replaced  by  a  strong  aqueous  solution  of  gentian  violet,  which  is 
poured  off  and  renewed  several  times  until  the  acid  has  been  replaced 
by  the  stain.  The  specimen  is  then  examined  in  the  coloring  solu- 
tion, after  soaking  up  the  excess  with  filter-paper,  the  thin  layer  of 
coloring  fluid  not  interfering  with  a  clear  view  of  the  bacteria  and 
their  capsules.  After  mounting  in  Canada  balsam  the  capsules 
are  not  nearly  so  distinct.  The  width  of  the  capsule  varies  from 
one-half  to  twice  the  thickness  of  the  bacillus.  Its  outer  margin  is 
stained,  leaving  a  clear  zone  immediately  about  the  bacillus. 

Cultivation. — The  bacillus  is  anaerobic  and  aerogenic.  It  grows 
upon  all  culture  media  at  the  room  temperature,  though  better  at 
the  temperature  of  incubation. 

Gelatin. — It  grows  in  ordinary  neutral  or  alkaline  gelatin,  but 
better  in  gelatin  containing  glucose,  in  which  the  characteristic  gas 
production  is  marked.  Soft  media,  made  with  5  instead  of  10 
per  cent,  of  the  crude  gelatin,  is  said  to  be  better  than  the  standard 
preparation. 

There  is  no  distinct  liquefaction  of  the  medium,  but  in  5  per  cent, 
gelatin  softening  can  sometimes  be  demonstrated  by  tilting  the  tube 
and  observing  that  the  gas  bubbles  change  their  position,  as  well  as 
by  noticing  that  the  growth  tends  to  sediment. 

Agar-agar. — In  making  agar-agar  cultures  careful  anaerobic  pre- 
cautions must  be  observed.  The  tubes  should  contain  considerably 
more  than  the  usual  quantity  of  the  medium,  which  should  be  boiled 
and  freshly  solidified  before  using.  The  implantation  should  be 
deeply  made  with  a  long  wire.  The  growth  takes  place  slowly  un- 
less such  tubes  are  placed  in  a  Buchner's  jar  or  other  anaerobic 
device.  The  deeper  colonies  are  the  largest.  Sometimes  the  growth 
takes  place  within  10-12  mm.  of  the  surface;  at  others,  within  3-4 
cm.  of  it.  After  repeated  cultivation  the  organisms  seem  to  become 


Cultivation 


335 


accustomed  to  the  presence  of  oxygen,  and  will  grow  higher  up  in 
the  tube  than  when  freshly  isolated. 

Colonies. — The  colonies  seen  in  the  culture-media  are  grayish- 
white  or  brownish-white  by  transmitted 
light,  and  sometimes  exhibit  a  central  dark 
dot.  At  the  end  of  twenty-four  hours  the 
larger  colonies  do  not  exceed  0.5-1.0  mm.  in 
diameter,  though  they  may  subsequently 
attain  a  diameter  of  2-3  mm.  or  more. 
Their  first  appearance  is  as  little  spheres  or 
ovals,  more  or  less  flattened,  with  irregular 
contours,  due  to  the  presence  of  small  pro- 
jecting prongs,  which  are  quite  distinct  under 
a  lens.  The  colonies  may  appear  as  little 
irregular  masses  with  projections. 

After  several  days  or  weeks,  single,  well- 
shaped  colonies  may  attain  a  large  size  and 
be  surrounded  by  projections,  either  in  the 
form  of  little  knobs  or  spikes  or  of  fine 
branchings — hair-like  or  feathery.  Their  ap- 
pearance has  been  compared  to  thistle-balls 
or  powder-puffs  and  to  thorn-apples.  When 
the  growth  takes  place  in  the  puncture,  the 
feathery  projections  are  continuous.  Bubbles 
of  gas  make  their  appearance  in  plain  agar  as 
well  as  in  sugar-agar,  though,  of  course,  less 
plentifully.  They  first  appear  in  the  line  of 
growth ;  afterward  throughout  the  agar,  often 
at  a  distance  from  the  actual  growth.  Any 
fluid  collecting  about  the  bubbles  or  at  the 
surface  of  the  agar-agar  may  be  turbid  from 
the  presence  of  bacilli.  The  gas-production 
is  more  abundant  at  37°C.  than  at  the  room 
temperature. 

The  agar-agar  is  not  liquefied  by  the  growth 
of  the  bacillus,  but  is  often  broken  up  into 
fragments  and  forced  into  the  upper  part  of 
the  tube  by  the  excessive  gas-production. 

Bouillon. — In  bouillon,  growth  does  not 
occur  in  tubes  exposed  to  the  air,  but  when 
the  tubes  are  placed  in  Buchner's  jars,  or 
kept  under  anaerobic  conditions,  it  occurs 
with  abundant  gas-formation,  especially  in 
glucose-bouillon,  and  the  formation  of  a 

frothy  layer  on  the  surface.     The  growth  is  rapid  in  development, 
the  bouillon  becoming  clouded  in  two  to  three  hours.     After  a  few 


•I 


Fig.  120. — Bacillus 
aerogenes  capsulatus, 
with  gas  production 
(from  photograph  by 
Prof.  Simon  Flexner). 


336  Gaseous  Edema 

days  the  bacilli  sediment  and  the  bouillon  again  becomes  clear.  The 
reaction  of  the  bouillon  becomes  strongly  acid. 

Milk. — In  milk  the  growth  is  rapid  and  luxuriant  under  anaerobic 
conditions,  but  does  not  take  place  in  cultures  exposed  to  the  air. 
The  milk  is  coagulated  in  from  twenty-four  to  forty-eight  hours, 
the  coagulum  being  either  uniform  or  firm,  retracted,  and  furrowed 
by  gas  bubbles.  When  litmus  has  been  added  to  the  milk,  it  be- 
comes decolorized  when  the  culture  is  kept  without  oxygen,  but  turns 
pink  when  it  is  exposed  to  the  air. 

Potato. — The  bacillus  will  also  grow  upon  potato  when  the  tubes 
are  inclosed  in  an  anaerobic  apparatus.  There  is  a  copious  gas- 
development  in  the  fluid  at  the  bottom  and  sides  of  the  tube,  so 
that  the  potato  becomes  surrounded  by  a  froth.  After  complete 
absorption  of  the  oxygen  a  thin,  moist,  grayish- white  growth  takes 
place  upon  the  surface  of  the  medium. 

Vital  Resistance. — The  vital  resistance  of  the  organism  is  not  great. 
Its  thermal  death-point  was  found  to  be  58°C.  after  ten  minutes' 
exposure.  Cultures  made  by  displacing  the  air  with  hydrogen  are 
less  vigorous  than  those  in  which  the  oxygen  is  absorbed  from  the 
air  by  pyrogallic  acid.  It  was  found  that  in  the  former  class  of 
cultures  the  bacillus  died  in  three  days,  while  in  the  absorption  ex- 
periments it  was  kept  alive  at  the  body  temperature  for  one  hundred 
and  twenty-three  days.  It  is  said  to  live  longer  in  plain  agar  than 
in  sugar-agar.  To  keep  the  cultures  alive  it  has  been  recommended 
to  seal  the  agar-agar  tube  after  two  or  three  days'  growth. 

Metabolic  Products. — The  bacillus  is  unable  to  make  use  of  the 
uncombined  oxygen  of  the  atmosphere,  and  derives  its  oxygen  sup- 
ply entirely  from  carbohydrates  in  the  medium  in  which  it  grows. 
It  causes  fermentation  of  most  carbohydrates  with  the  evolution  of 
much  gas  and  some  acid.  It  coagulates  milk. 

Simonds*  divides  the  organisms  known  as  B.  aerogenes  capsulatus 
or  B.  welchii  into  four  groups  according  to  their  metabolic  activities 
as  follows: 

1.  Organisms  that  ferment  inulin  and  glycerin  with  production 
of  gas  and  increase  of  acidity.     Do  not  form  spores  in  media  con- 
taining  either    substance.     Produce    strong    hemolysins,   and   are 
pathogenic  for  guinea-pigs,   even  after  many  months  cultivation 
upon  artificial  media. 

2.  Organisms  that  produce  acid  and  gas  from  glycerin  but  not 
from  inulin.     Form   spores  in  inulin  but  not  in  glycerin  broth. 
Hemolytic  and  pathogenic  powers  variable. 

3.  Organisms  that  produce  acid  and  gas  from  inulin  but  not  from 
glycerin.     Form  spores  in  glycerin  but  not  in  inulin  broth.     Hem- 
olysis  and  pathogenicity  variable. 

4.  Organisms  that  do  not  produce  acid  or  gas  from  either  inulin 
or  glycerin  and  from  spores  in  both  inulin  and  glycerin  broths. 

*Jour.  Infectious  Diseases,  1915,  xvi,  32. 


Pathogenesis  337 

Pathogenesis. — The  pathogenic  powers  of  the  bacillus  are  limited, 
and  while  in  some  infected  cases  it  seems  to  be  the  cause  of  death, 
its  power  to  do  mischief  in  the  body  seems  to  depend  entirely  upon 
the  pre-existence  of  depressing  and  devitalizing  conditions  predis- 
posing to  its  growth. 

Being  anaerobic,  the  bacilli  are  unable  to  live  in  the  circulating 
blood,  though  they  grow  in  old  clots  and  in  cavities,  such  as  the 
uterus,  etc.,  where  little  oxygen  enters,  and  from  which  they  enter 
the  blood  and  are  distributed. 

In  support  of  these  views  Welch  and  Nuttall  show  that  when  2.5 
cc.  of  a  fresh  sugar-bouillon  culture  are  injected  into  the  ear- vein  of 
a  healthy  rabbit,  it  usually  recovers.  After  similar  injection  with 
but  i  cc.  of  the  culture,  a  pregnant  rabbit  carrying  two  dead  embryos, 
died  in  twenty-one  hours.  It  seems  that  the  bacilli  were  first  able 
to  secure  a  foothold  in  the  dead  embryos,  and  there  multiplied  suffi- 
ciently to  bring  about  the  subsequent  death  of  the  mother. 

After  death,  when  the  blood  is  no  longer  oxygenated,  the  bacilli 
grow  rapidly,  with  marked  gas-production,  which  in  some  cases  is 
said  to  cause  the  body  to  swell  to  twice  its  natural  size.  The  effect 
upon  guinea-pigs  does  not  differ  from  that  upon  rabbits,  though 
gaseous  phlegmons  are  sometimes  produced. 

Pigeons,  when  subcutaneously  inoculated  in  the  pectoral  region, 
frequently  die  in  from  seven  to  twenty-four  hours,  but  may  recover. 
Gas-production  causes  the  tissues  to  become  emphysematous. 

Intraperitoneal  inoculation  sometimes  causes  fatal  purulent  peri- 
tonitis of  laboratory  animals. 

Sources  of  Infection. — The  infection  seen  in  man  usually  occurs 
from  wounds  into  which  earth  has  been  ground,  as  in  the  case  of  a 
compound,  comminuted  fracture  of  the  humerus,  with  fatal  infec- 
tion, reported  by  Dunham,  or  in  wounds  and  injuries  in  the  neigh- 
borhood of  the  perineum. 

Among  the  twenty- three  cases  reported  by  Welch  and  Flexner* 
we  find  wounds  of  the  knee,  leg,  hip,  and  forearm,  ulcer  of  the 
stomach,  typhoid  ulcerations  of  the  intestine,  strangulated  hernia 
with  operation,  gastric  and  duodenal  ulcer,  perineal  section,  and 
aneurysm,  as  conditions  in  which  external  or  gastro-intestinal  in- 
fection occurred. 

Dobbin, t  P.  Ernst, {  Graham,  Stewart  and  Baldwin, §  and  Kronig 
and  Menge||  have  studied  cases  of  puerperal  sepsis  and  sepsis  follow- 
ing abortion  either  caused  by  the  bacillus  or  in  which  it  played  an 
important  role. 

Williams**  has  found  the  bacillus  in  a  case  of  suppurative  pyelitis. 

"  "Journal  of  Experimental  Medicine,"  Jan.,  1896,  vol.  i,  No.  i. 
t  ''Bull.  Johns  Hopkins  Hospital,"  Feb.,  1897,  No.  71,  p.  24. 

"Virchow's  Archiv,"  Bd.  cxxxm,  Heft  2. 

"Columbus  Med.  Jour.,"  Aug.,  1893. 

"  Bakteriologie  des  weiblichen  Genitalkanals,"  Leipzig,  1897. 

"Bull.  Johns  Hopkins  Hospital,"  April,  1896,  p.  66. 


338 


Gaseous  Edema 


The  symptoms  following  infection  are  quite  uniform,  consisting 
of  redness  and  swelling  of  the  wound,  with  rapid  elevation  of  tem- 
perature and  rapid  pulse.  The  wound  usually  becomes  more  or 
less  emphysematous,  and  discharges  a  thin,  dirty,  brownish,  offensive 
fluid  that  contains  gas  bubbles  and  is  sometimes  frothy.  The  pa- 
tients occasionally  recover,  especially  when  the  infected  part  can 
be  amputated,  but  death  is  the  common  outcome.  After  death  the 
body  begins  to  swell  almost  immediately,  may  attain  twice  its 
normal  size  and  be  unrecognizable.  Upon  palpation  a  peculiar  crepi- 
tation can  be  felt  in  the  subcutaneous  tissue  nearly  everywhere, 
and  the  presence  of  gas  in  the  blood-vessels  is  easy  of  demonstra- 
tion. The  gas  is  inflammable,  and  as  the  bubbles  ignite  explosive 
sounds  are  heard. 


Fig.    121. — "Frothy    liver"    from    Bacillus    aerogenes     capsulatus     infection 

(Aschoff). 

At  the  autopsy  the  gas  bubbles  are  found  in  most  of  the  internal 
organs,  sometimes  so  numerously  as  to  justify  the  German  term 
" Schaumorgane "  (frothy  organs).  The  liver  is  especially  apt  to 
show  this  condition  When  such  tissues  are  hardened  and  ex- 
amined microscopically,  the  bubbles  appear  as  spaces  in  the  tissue, 
their  borders  lined  with  large  numbers  of  the  bacillus.  There  are 
also  clumps  of  bacilli  without  gas  bubbles,  but  surrounded  by 
tissue,  whose  nuclei  show  a  disposition  to  fragment  or  disappear, 
and  whose  cells  and  fibers  show  signs  of  disintegration  and  fatty 
change.  In  discussing  these  changes  Ernst  concluded  that  they 
were  ante-mortem  and  due  to  the  irritation  caused  by  the  bacillus. 
The  gas-production  he  regards  as  post-mortem. 

In  the  internal  organs  the  bacillus  is  usually  found  in  pure  culture, 
but  in  the  wound  it  is  usually  mixed  with  other  bacteria.  On  this 
account  it  is  difficult  to  estimate  just  how  much  of  the  damage  be- 
fore death  depends  upon  the  activity  of  the  gas  bacillus.  That 


Pathogenesis  339 

gas-production  after  death  has  nothing  to  do  with  pathogenesis 
during  life  is  shown  by  injecting  into  the  ear-vein  of  a  rabbit  a 
liquid  culture  of  the  gas  bacillus,  permitting  about  five  minutes' 
time  for  the  distribution  of  the  bacilli  throughout  the  circula- 
tion, and  then  killing  the  rabbit.  In  a  few  hours  the  rabbit  will 
swell  and  its  organs  and  tissues  be  riddled  with  the  gas  bubbles. 

At  times,  however,  as  in  a  case  of  Graham,  Stewart  and  Baldwin, 
there  is  no  doubt  but  that  the  bacillus  produces  gas  in  the  tissues 
of  the  body  during  life.  These  observers,  in  a  case  of  abortion  with 
subsequent  infection,  found  the  patient  "  emphysematous  from  the 
top  of  her  head  to  the  soles  of  her  feet"  several  hours  before  death. 

In  this  case,  in  which  the  bacillus  was  found  in  pure  culture, 
it  would  indeed  be  difficult  to  doubt  that  the  fatal  issue  was  due  to 
Bacillus  aerogenes  capsulatus. 

An  excellent  review  of  the  early  literature  of  the  subject  is  to  be 
found  in  "  A  Contribution  to  the  Knowledge  of  the  Bacillus  Aerog- 
enes Capsulatus,"  by  W.  T.  Howard,  Jr.* 

*  "  Contributions  to  the  Science  of  Medicine  by  the  Pupils  of  W.  H.  Welch," 
1900,  p.  461. 


CHAPTER  III 
TETANUS 

BACILLUS  TETANI  (FLUGGE) 

General  Characteristics. — A  motile,  flagellated,  sporogenous,  liquefying, 
obligatory  anaerobic,  non-chromogenic,  aerogenic,  toxic,  pathogenic  bacillus  of 
the  soil,  staining  by  ordinary  methods  and  by  Gram's  method.  Its  chief 
morphologic  characteristic  is  the  occurrence  of  a  large  round  spore  at  one  end. 

The  bacillus  of  tetanus  was  discovered  by  Nicolaier*  in  1884, 
and  obtained  in  pure  culture  by  Kitasatof  in  1889.  It  is  universally 
acknowledged  to  be  the  cause  of  tetanus  or  "lock-jaw." 

Distribution. — The  tetanus  bacillus  is  a  common  saprophyte  in 
garden  earth,  dust,  and  manure,  and  is  a  constant  parasite  in  the  in- 
testinal contents  of  herbivorous  animals. 

The  relation  of  the  bacillus  to  manure  is  interesting,  but  it  is  most 
probable  that  manured  ground,  because  it  is  richer,  permits  the 
bacilli  to  flourish  better  than  sterile  ground.  The  common  occur- 
rence of  the  bacilli  in  the  excrement  of  herbivorous  animals  is  to  be 
explained  through  the  accidental  ingestion  of  earth  with  the  food 
cropped  from  the  ground.  The  spores  of  the  bacillus  thus  reaching 
the  intestine  seem  able  to  develop  because  of  appropriate  anaerobic 
conditions.  Verneuil  has  observed  that  tetanus  rarely  occurs  at  sea 
except  upon  cattle  transports. 

Le  Dantec|  has  shown  that  the  tetanus  bacillus  is  a  common  or- 
ganism in  New  Hebrides,  where  the  natives  poison  their  arrows 
by  dipping  them  into  a  clay  rich  in  its  spores. 

Morphology. — The  tetanus  bacillus  is  a  long,  slender  organism 
measuring  0.3  to  0.5  X  2  to  4  /*  (Fliigge).  Its  most  striking  char- 
acteristic is  an  enlargement  of  one  end,  which  contains  a  large  round 
spore.  The  bacilli  in  which  no  spores  are  yet  formed  have  rounded 
ends  and  seldom  unite  in  chains  or  pairs.  They  are  motile  and 
have  many  flagella  arising  from  all  parts  of  the  surface  (petrichia). 

Staining. — The  bacilli  stain  readily  with  ordinary  aqueous  solu- 
tions of  the  anilin  dyes  and  by  Gram's  method. 

Isolation. — The  method  usually  employed  for  the  isolation  of  the 
tetanus  bacillus  was  originated  by  Kitasato,  and  based  upon  the 
observation  that  its  spores  can  resist  exposure  to  high  temperatures 
for  considerable  periods  of  time.  After  rinding  by  microscopic 
examination  that  the  bacilli  were  present  in  pus,  Kitasato  spread  it 
upon  the  surface  of  an  ordinary  agar-agar  tube  and  incubated  it  for 

*  "Deutsche  med.  Wochenschrift,"  1884,  42. 

flbid.,  1889,  No.  31. 

j  See  attracts  in  the  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  rx,  286;  xm,  351. 

340 


Isolation 


34i 


Fig.  122.— Bacillus  tetani.      X  1000  (Frankel  and  Pfeiffer). 


Fig.  123.— Bacillus  tetani;  six-day-  Fig.  124.— Bacillus  tetani;  culture 
old  puncture  culture  in  glucose-gelatin  four  days  old  in  glucose-gelatin  (Frankel 
(Frankel  and  Pfeiffer).  and  Pfeiffer). 


342  Tetanus 

twenty-four  hours,  during  which  time  all  of  the  contained  micro- 
organisms, including  the  tetanus  bacillus,  increased  in  number. 
He  then  exposed  it  for  an  hour  to  a  temperature  of  8o°C.,  by  which 
all  fully  developed  bacteria,  tetanus  as  well  as  the  others,  and  the 
great  majority  of  the  spores,  were  destroyed.  As  scarcely  anything 
but  the  tetanus  spores  remained  alive,  their  subsequent  growth 
gave  a  fairly  pure  culture. 

Cultivation. — The  tetanus  bacillus  is  difficult  to  cultivate  because 
it  will  not  grow  where  the  smallest  amount  of  free  oxygen  is  present. 
It  is  hence  a  typical  obligatory  anaerobe.  Farran*  and  Grixoni 


Fig.  125. — Bacillus  tetani;  five-day-old  colony  upon  gelatin  containing  glucose. 
X    1000    (Frankel    and    Pfeiffer). 

believe  it  to  have  originally  been  an  optional  anaerobe,  and  it  is  said 
by  these  writers  that  the  organism  can  gradually  be  accustomed  to 
oxygen  so  as  to  grow  in  its  presence.  When  this  is  achieved,  it  loses 
its  virulence. 

The  general  methods  for  the  cultivation  of  anaerobic  organisms, 
are  given  under  the  appropriate  heading  (Anaerobic  Cultures),  and 
need  not  be  repeated  here. 

The  colonies  of  the  tetanus  bacillus,  when  grown  upon  gelatin 
plates  in  an  atmosphere  of  hydrogen,  resemble  those  of  the  well- 
known  hay  bacillus.  There  is  a  rather  dense,  opaque  central  mass 
surrounded  by  a  more  transparent  zone,  the  margins  of  which  con- 
sist of  a  fringe  of  radially  projecting  bacilli.  Liquefaction  occurs 
slowly. 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.."  July  15,  1898,  p.  28. 


Vital  Resistance 


343 


Gelatin. — The  growth  occurs  deep  in  the  puncture,  and  is  arbores- 
cent. Liquefaction  begins  in  the  second  week  and  causes  the  dis- 
appearance of  the  radiating  filaments.  The  liquefaction  spreads 
slowly,  but  may  involve  the  entire  mass  of 
gelatin  and  resolve  it  into  a  grayish-white 
syrupy  liquid,  at  the  bottom  of  which  the 
bacilli  accumulate.  The  growth  in  gelatin 
containing  glucose  is  rapid. 

Agar-agar. — The  growth  in  agar-agar 
punctures  is  slower,  but  similar  to  the  gela- 
tin cultures  except  for  the  absence  of 
liquefaction. 

Bouillon. — The  organism  can  be  grown 
in  bouillon  without  difficulty,  when  once 
habituated  to  the  medium.  The  bouillon 
should  be  heated  to  drive  off  the  air,  then 
rapidly  cooled  and  the  transplantation 
made.  If  there  be  a  depth  of  10  cm.  the 
bacilli  grow  readily  in  the  lower  half  of  the 
fluid.  If  the  surface  be  covered  with  liquid 
paraffin  before  the  final  sterilization  and  in- 
oculation, they  grow  throughout  the  entire 
medium.  The  organism  attains  its  maxi- 
mum development  at  a  temperature  of 
37°C.  Gas  is  given  off  from  the  cultures, 
and  they  have  a  peculiar  odor,  very  char- 
acteristic, but  difficult  to  describe.  The 
bouillon  is  clouded  and  contains  a  sediment. 

In  bouillon  containing  sugar  considerable 
gas  is  formed  in  the  fermentation  tube. 
Both  CO2  and  H2S  are  formed. 

Milk  is  favorable  for  the  development  of 
the  tetanus  bacillus.  There  is  no  coagula- 
tion. Litmus  milk  is  acidified. 

Potato. — Upon  potatoes  under  strict  anae- 
robic conditions  the  bacilli  grow  but  slightly. 

Vital  Resistance. — The  tetanus  spores 
may  remain  alive  in  dry  earth  for  many 
years.  Sternberg  says  they  can  resist  im- 
mersion in  5  per  cent,  aqueous  carbolic  acid 
solutions  for  ten  hours,  but  fail  to  grow  after 
fifteen  hours.  A  5  per  cent,  carbolic  acid 
solution,  to  which  0.5  per  cent,  of  hydro- 
chloric acid  has  been  added,  destroys  them 
in  two  hours.  They  are  destroyed  in  three  hours  by  i  :  1000  bi- 
chlorid  of  mercury  solution,  but  when  to  such  a  solution  0.5  per  cent, 
of  hydrochloric  acid  is  added,  its  activity  is  so  increased  that  the 


Fig.  126. — Tetanus 
bacillus;  glucose-agar 
culture,  five  months  old 
(Curtis). 


344  Tetanus 

spores  are  destroyed  in  thirty  minutes.  According  to  Kitasato,* 
exposure  to  streaming  steam  for  from  five  to  eight  minutes  is 
certain  to  kill  tetanus  spores,  and  this  statement  has  found  its  way 
into  most  of  the  text-books  without  discussion.  Theobald  Smith,  f 
however,  has  studied  several  cultures  of  the  organism  and  finds  that 
its  resistance  to  heat  is  much  greater,  and  that  in  one  case  seventy 
minutes'  exposure  to  streaming  steam  did  not  kill  all  of  the  spores. 

Metabolic  Products. — Bouillon  cultures  of  the  tetanus  bacillus 
contain  acids,  proteolytic  ferment,  and  several  toxic  substances, 
of  which  tetanospasmin  and  tetanolysin  are  best  known.  The 
toxic  products  are  apparently  all  soluble.  No  endotoxin  is  known  to 
be  formed. 

The  most  ready  method  of  preparing  the  toxins  for  experimental 
study  is  to  cultivate  the  bacilli  in  freshly  prepared  neutral  or  slightly 
alkaline  sugar-free  bouillon  under  conditions  of  most  strict  anaero- 
biosis,  at  a  temperature  of  37°C.,  and  then  filter  the  culture  through 
porcelain.  FieldJ  found  the  highest  degree  of  toxicity  about  the 
sixth  or  seventh  day.  It  may  attain  a  toxicity  so  great  that  0.000005 
c.c.  will  cause  the  death  of  a  mouse.  The  average  culture  has  such 
toxicity  that  o.ooi  c.c.  is  fatal  to  a  guinea-pig.  Knorr§  gives  some 
interesting  comparisons  of  the  susceptibility  of  different  animals,  as 
follows: 

gram  of  horse  is  destroyed  by x  toxin 

gram  of  goat  is  destroyed  by 2X  toxin 

gram  of  mouse  is  destroyed  by..  .  .  : i$x  toxin 

gram  of  rabbit  is  destroyed  by 2,ooo#  toxin 

gram  of  hen  is  destroyed  by 200,000:*;  toxin 

The  toxin  is  very  unstable,  and  is  easily  destroyed  by  heat  above 
6o°C.  It  is  also  quickly  destroyed  by  light,  especially  direct  sun- 
light. Flexner  and  Noguchi||  found  that  5  per  cent,  of  eosin  added 
to  the  toxin  destroyed  it  through  the  photodynamic  power  of  the 
stain.  It  is  also  easily  destroyed  by  electric  currents.  The  best 
method  of  keeping  it  is  to  add  0.5  per  cent,  of  phenol,  and  then  store 
it  in  a  cool,  dark  place,  in  bottles  completely  filled  and  tightly  corked. 
It  will  not  keep  its  strength  in  liquid  form  under  the  best  conditions. 

To  keep  it  for  experimental  purposes  it  is  advisable  to  precipitate 
the  toxin  from  the  bouillon  by  supersaturation  with  ammonium  sul- 
phate, which  causes  it  to  float  upon  the  liquid  in  the  form  of  a  sticky 
brown  scum  that  can  be  skimmed  off  and  dried.  Such  dry  precipi- 
tate retains  its  activity  for  months. 

From  cultures  of  tetanus  bacilli  grown  in  various  media,  and  from 
the  blood  and  tissues  of  animals  affected  with  the  disease,  Brieger 
succeeded  in  separating  "tetanin,"  "tetanotoxin,"  tetanospasmin," 
and  a  fourth  substance  to  which  no  name  is  given.  All  were  very 

*  "Zeitschrift  fur  Hygiene,"  xn,  p.  225. 

t  "Jour.  Amer.  Med.  Assoc.,"  March  21,  1908,  vol.  L,  No.  12,  p.  931. 

J  "-Proc.  N.  Y.  Path.  Soc,"  March,  1904,  p.  18. 

§  "Munch,  med.  Wochenschrift,"  1898,  p.  321. 

I!  "Studies  from  the  Rockefeller  Institute,"  1905,  v. 


Metabolic  Products  345 

poisonous  and  productive  of  tonic  convulsions.  Later  Brieger  and 
Frankel  isolated  an  extremely  poisonous  toxalbumin  from  sugar- 
bouillon  cultures  of  the  bacillus.  Ehrlich*  later  discovered  a  new 
poisonous  element  to  which  he  applied  the  name  tetanolysin. 

The  purified  toxin  of  Brieger  and  Cohn  was  fatal  to  mice  in  doses 
of  0.00000005  gram.  Lambertf  considers  the  tetanus  toxin  to  be 
the  most  poisonous  substance  that  has  ever  been  discovered. 

Fermi  and  PernossJ  found  most  toxin  produced  in  agar-agar 
cultures,  less  in  gelatin  cultures,  and  least  in  bouillon  cultures. 

Ehrlich§  found  two  poisons  in  the  tetanus  toxin,  one  of  which 
was  convulsive  and  was  in  consequence  called  tetanospasmin,  the 
other  hemolytic  and  called  tetanolysin.  When  tetanus  toxin  is 
added  to  defibrinated  blood,  the  tetanolysin  is  absorbed  by  the 
corpuscles,  many  of  which  are  dissolved,  while  the  tetanospasmin 
remains  unchanged. 

Donitz||  and  Wassermann  and  Takaki**  have  found  that  the 
tetanus  toxin  has  a  specific  affinity  for  the  central  nervous  system, 
with  whose  cells  it  combines  in  vitro  and  becomes  inert. 

Roux  and  Borrelff  have  found  that  when  tetanus  toxin  is  injected 
into  the  brain  substance  a  very  much  smaller  dose  will  cause  death 
than  is  necessary  when  the  poison  is  absorbed  from  the  subcutaneous 
tissues. 

Like  most  of  the  bacterial  toxins,  the  tetanus  poison  is  only  effect- 
ive when  produced  in  or  injected  into  the  tissues  and  absorbed  into 
the  circulation.  It  is  harmless  when  given  by  the  digestive  tract, 
RamonJt  having  administered  by  the  mouth  300,000  times  the  fatal 
hypodermic  dose  without  producing  any  symptoms. 

One  of  the  most  interesting  peculiarities  about  the  toxin  is  the  com- 
parative uniformity  of  the  period  intervening  between  its  administra- 
tion and  the  appearance  of  the  symptoms — erroneously  called  the 
incubation  period.  This  varies  within  a  narrow  margin,  inversely, 
with  the  size  of  the  dose.  Thus,  according  to  Behring,  the  effect  of 
varying  doses  of  the  toxin  upon  mice  becomes  evident  according  to 
the  size  of  the  dose  in  from  twelve  to  thirty-six  hours,  thus: 

13  lethal  doses symptoms  in  36  hours 

no  lethal  doses symptoms  in  24  hours 

333  lethal  doses symptoms  in  20  hours 

1300  lethal  doses symptoms  in  14  hours 

3600  lethal  doses symptoms  in  12  hours 

The  local  action  of  the  toxin  is  very  painful  and  associated  with 
spasm  of  the  muscular  fibers  with  which  it  comes  in  contact.  Pit- 

*  "Berliner  klin.  Wochenschrift,"  1898. 

t  "New  York  Med.  Jour.,"  June  5,  1897. 

j  "Centralbl.  f.  Bakt.,"  etc.,  xv,  p.  303. 

§  "Berliner  klin.  Wochenschrift,"  1898,  No.  12,  p.  273. 

||  "Deutsche  med.  Wochenschrift,"  1897,  p.  428. 
**  "Berliner  klin.  Wochenschrift,"  1898,  35. 
ft  "Ann.  de  1'Inst.  Pasteur,"  1898  xn. 
it  "Deutsche  med.  Wochenschrift,"  Feb.  24,  1898. 


346  Tetanus 

field,*  thinking  that  it  might  be  useful  in  the  treatment  of  certain 
paralytic  affections,  injected  a  minute  quantity  of  it  into  the  calf 
of  his  leg  and  experienced  the  severe  spasmodic  local  effects  of  the 
poison  for  twelve  hours. 

It  has  been  the  belief  of  most  physiologists  that  tetanus  toxin 
acts  solely  upon  the  motor  cells  of  the  spinal  cord,  and  causes  the 
tonic  spasms  as  strychnin  does.  The  affinity  of  the  toxin  for  the 
nervous  tissues  has  been  made  the  subject  of  careful  investigations  by 
Marie  and  Moraxf  and  Meyer  and  Ransom. J  The  former  found 
that  the  absorption  of  tetanus  toxin  took  place  partly  through  the 
peripheral  nerves  because  of  specific  affinity  between  the  toxin  and 
the  axis  cylinder  substance;  the  latter  found  the  toxin  carried  to  the 
central  nervous  system  solely  by  the  motor  nerves,  the  action  depend- 
ing upon  the  integrity  of  the  axis  cylinder.  They  believe  that  the 
toxin  is  absorbed  by  the  axis  cylinder  endings,  and  reaching  the  cor- 
responding spinal  nerve  center  by  that  route  spreads  to  the  corre- 
sponding center  in  the  other  half  of  the  cord  and  outward,  resulting 
in  generalized  tetanus.  When  intoxication  is  produced  through  the 
circulation,  the  poison  is  taken  up  by  the  nerve  endings  in  all  parts 
of  the  body,  and  the  disease  is  not  localized,  but  general.  Antitoxin, 
unlike  the  toxin,  does  not  travel  by  the  nerve  route,  but  is  found  only 
in  the  blood  and  lymph.  Zupnik§  has  brought  forward  evidence 
that  this  view  is  incorrect  and  that  there  are  two  distinct  actions 
caused  by  the  toxin.  He  differentiates  between  tetanus  ascendens 
and  tetanus  descendens.  The  former  always  follows  the  intramus- 
cular introduction  of  the  toxin,  and  depends  upon  its  direct  action 
upon  the  muscle  itself.  It  explains  the  familiar  phenomenon  of 
rigidity  making  its  first  appearance  in  that  member  into  which  the 
inoculation  was  made.  The  ascending  tetanus  gradually  ascends 
from  muscle  to  muscle.  He  thinks  the  absorption  of  the  poison  by 
the  muscle-cells  depends  upon  their  normal  metabolic  function,  as 
when  their  nerves  are  severed,  the  fixation  of  the  toxin  and  the 
occurrence  of  the  tonic  spasm  does  not  occur. 

Tetanus  descendens  results  from  the  entrance  of  the  toxin  into  the 
circulation  from  the  cellular  tissue  and  its  distribution  in  the  blood. 
Under  these  conditions  Zupnik  believes  it  acts  upon  the  central 
nervous  system,  especially  upon  the  spinal  cord,  manifesting  itself 
in  extreme  reflex  excitability  with  irregular  motor  discharges  result- 
ing in  clonic  spasms. 

There  are,  therefore,  two  forms  of  spasm  in  tetanus:  the  tonic 
convulsions,  seeming  to  depend  upon  local  action  and  fixation  of  the 
toxin,  and  the  clonic  convulsions,  depending  upon  the  centric  action. 
The  latter  are  the  more  dangerous  for  the  sufferer. 

*  "Therapeutic  Gazette,"  March  15,  1897. 

t"Ann  de  1'Inst.  Pasteur,"  1902,  xvi,  p.  818;  and  "Bull,  de  1'Inst.  Past.," 
1903,  i,  p.  41. 

t  "Arch.  f.  exper.  Path.  u.  Pharmak.,"  1903,  XLIX. 
§  "Wiener  klin.  Wochenschrift,"  Jan.  23,  1902. 


Pathogenesis  347 

The  lockjaw  or  trismus  and  the  opisthotonos  that  are  so  charac- 
teristic of  the  affection  depend,  according  to  Zupnik's  view,  upon  a 
loss  of  equilibrium  among  the  muscles  affected.  They  occur  only 
in  descending  tetanus  and  depend  upon  spasm  of  muscles  without 
equally  powerful  opposing  groups.  The  stronger  muscles  of  the  jaw 
are  those  that  close  it;  the  stronger  muscles  of  the  back,  those  of  the 
erector  group.  This  view  is  exactly  the  opposite  of  Meyer  and  Ran- 
som,* who  believe  that  the  tetanus  toxin  is  absorbed  only  along  the 
nerve  trunks,  and  found  that  section  of  the  spinal  cord  prevented 
the  ascent  of  tetanus  from  the  lower  extremities.  Injection  of  the 
toxin  into  a  posterior  nerve-root  produced  tetanus  dolorosus.  In- 
jection of  the  toxin  into  a  posterior  nerve-root  together  with  section 
of  the  spinal  cord  produced  exaltation  of  the  reflex  irritability — 
"  Jactationstetanus."  Injection  in  sensory  nerves  does  not  produce 
tetanus  dolorosus  because  the  transportation  of  the  poison  along 
these  trunks  is  so  slow. 

The  tetanolysin  is  a  hemolytic  component  of  the  toxic  bouillon, 
and  is  entirely  separate  and  distinct  from  the  tetanospasmin  or  con- 
vulsive poison.  It  probably  takes  no  part  in  the  usual  clinical 
manifestations  of  tetanus. 

Pathogenesis. — The  work  of  Kitasato  has  given  us  very  complete 
knowledge  of  the  biology  of  the  tetanus  bacillus  and  completely 
established  its  specific  nature. 

When  a  white  mouse  is  inoculated  with  an  almost  infinitesimal 
amount  of  tetanus  culture,  or  with  garden  earth  containing  the  tet- 
anus bacillus,  the  first  symptoms  come  on  in  from  one  to  two  days, 
when  the  mouse  develops  typical  tetanic  convulsions,  first  beginning 
in  the  neighborhood  of  the  inoculation,  but  soon  becoming  general. 
Death  follows  sometimes  in  a  very  few  hours.  In  rabbits,  guinea- 
pigs,  mice,  rats,  and  other  small  animals  the  period  of  incubation  is 
from  one  to  three  days.  In  man  the  period  of  incubation  varies 
from  a  few  days  to  several  weeks,  and  averages  about  nine  days. 

The  disease  is  of  much  interest  because  of  its  purely  toxic  nature. 
There  is  usually  a  small  wound  with  a  slight  amount  of  suppuration 
and  at  the  autopsy  the  organs  of  the  body  are  normal  in  appearance, 
except  the  nervous  system,  which  bears  the  greatest  insult.  It, 
however,  shows  little  else  than  congestion  either  macroscopically  or 
microscopically. 

The  conditions  in  the  animal  body  are  in  general  unfavorable  to 
the  development  of  the  bacilli,  because  of  the  loosely  combined 
oxygen  contained  in  the  blood,  and  they  grow  with  great  slowness, 
remaining  localized  at  the  seat  of  inoculation,  and  never  entering  the 
blood.  Doubtless  most  cases  of  tetanus  are  mixed  infections  in 
which  the  bacillus  enters  with  aerobic  bacteria,  that  aid  its  growth 
by  absorbing  the  oxygen  in  the  neighborhood.  The  amount  of 
poison  produced  must  be  exceedingly  small  and  its  power  tremen- 
*  "Archiv.  f.  exper.  Path.  u.  Pharmak.,"  1903,  Bd.  XLIX,  p.  396- 


348  Tetanus 

dous,  else  so  few  bacilli  growing  under  adverse  conditions  could  not 
produce  fatal  toxemia.  The  toxin  is  produced  rapidly,  for  Kitasato 
found  that  if  mice  were  inoculated  at  the  root  of  the  tail,  and  the  skin 
and  the  subcutaneous  tissues  around  the  inoculation  afterward 
either  excised  or  burned  out,  the  treatment  would  not  save  the  ani- 
mal unless  the  operation  were  performed  within  an  hour  after  the 
inoculation. 

Some  incline  to  the  view  that  the  toxin  is  a  ferment,  and  the 
experiments  of  Nocard*  might  be  adduced  in  support  of  the  theory. 
He  says:  "Take  three  sheep  with  normal  tails,  and  insert  under  the 
skin  at  the  end  of  each  tail  a  splinter  of  wood  covered  with  the  dried 
spores  of  the  tetanus  bacillus;  watch  these  animals  carefully  for  the 
first  symptoms  of  tetanus,  then  amputate  the  tails  of  two  of  them  20 
cm.  above  the  point  of  inoculation,  .  .  .  the  three  animals  succumb 
to  the  disease  without  showing  any  sensible  difference." 

The  circulating  blood  of  diseased  animals  is  fatal  when  injected 
into  susceptible  animals  because  of  the  toxin  it  contains;  and  the 
fact  that  the  urine  is  also  toxic  to  mice  proves  that  the  toxin  is  ex- 
creted by  the  kidneys. 

Two  classes  of  infected  wounds  are  particularly  apt  to  be  followed 
by  tetanus — namely,  those  into  which  soil  has  been  carried  by  the 
injuring  implement  and  those  of  considerable  depth.  The  infecting 
organism  reaches  the  first  class  in  large  numbers,  but  finds  itself 
under  aerobic  and  other  inappropriate  conditions  of  growth.  It 
reaches  the  second  class  in  smaller  numbers,  but  finds  the  conditions 
of  growth  better  because  of  the  depth  of  the  wound. 

The  severity  of  the  wound  has  nothing  whatever  to  do  with  the 
occurrence  of  tetanus,  pin-pricks,  nail  punctures,  insect  stings, 
vaccination,  and  a  variety  of  other  mild  injuries  sometimes  being 
followed  by  it. 

An  interesting  fact  has  been  presented  by  Vaillard  and  Rouget,f 
who  found  that  if  the  tetanus  spores  were  introduced  into  the  body 
freed  from  their  poison,  they  were  unable  to  produce  the  disease 
because  of  the  promptness  with  which  the  phagocytes  took  them  up. 
If,  however,  the  toxin  was  not  removed,  or  if  the  body-cells  were 
injured  by  the  simultaneous  introduction  of  lactic  acid  or  other 
chemic  agent,  the  spores  would  immediately  develop  into  bacilli, 
begin  to  manifacture  toxin,  and  produce  the  disease.  This  suggests 
that  many  wounds  may  be  infected  by  the  tetanus  bacillus  though 
the  surrounding  conditions  rarely  enable  it  to  develop  satisfactorily 
and  produce  enough  toxin  to  cause  disease. 

In  very  rare  cases  tetanus  may  possibly  occur  without  the  pre- 
vious existence  of  a  wound,  as  in  the  case  reported  by  Kamen,  who 
found  the  intestine  of  a  person  dead  of  the  disease  rich  in  Bacillus 
tetani.  Kamen  is  of  the  opinion  that  the  bacilli  can  grow  in  the 

*  Quoted  before  the  Academic  de  Medicine,  Oct.  22,  1895. 

t  See  "  Centralbl.  f .  Bakt.  Infekt.,  u.  Parasitenk.,"  vol.  xvi,  p.  208. 


Antitoxin  of  Tetanus  349 

intestine  and  be  absorbed,  especially  where  imperfections  in  the 
mucosa  exist. 

Montesano  and  Montesson,*  unexpectedly  found  the  tetanus 
bacillus  in  pure  culture  in  the  cerebro-spinal  fluid  of  a  case  of  para- 
lytic dementia  that  died  without  a  tetanic  symptom. 

Immunity. — All  animals  are  not  alike  susceptible  to  tetanus. 
Men,  horses,  mice,  rabbits,  and  guinea-pigs  are  susceptible;  dogs 
much  less  so.  Cattle  suffer  chiefly  after  castration,  accouchement, 
or  abortion.  Most  birds  are  scarcely  at  all  susceptible  either  to  the 
bacilli  or  to  their  toxin.  Amphibians  and  reptiles  are  immune, 
though  it  is  said  that  frogs  can  be  made  susceptible  by  elevation  of 
their  body-temperature. 

The  injection  of  the  toxic  bouillon  or  of  the  redissolved  ammonium 
sulphate  precipitate,  in  progressively  increasing  doses,  into  animals, 
causes  the  formation  of  antibodies  (antitoxin)  by  which  the  effects  of 
both  the  tetanospasmin  and  the  tetanolysin  are  destroyed.  The 
purely  toxic  character  of  the  disease  makes  it  peculiarly  well  adapted 
for  treatment  with  antitoxin,  and  at  the  present  time  our  sole 
therapeutic  reliance  is  placed  upon  it.  The  mode  of  preparing  the 
serum  and  the  system  of  standardization  are  discussed  in  the  section 
upon  Antitoxins  in  the  part  of  this  work  that  treats  of  the  Special 
Phenomena  of  Infection  and  Immunity. 

Antitoxin. — Numerous  cases  of  the  beneficial  action  of  antitoxin 
are  on  record,  but,  as  Welchf  has  pointed  out,  the  antitoxin  of 
tetanus  is  a  disappointment  in  the  treatment  of  tetanus.  Moschco- 
witz,{  in  his  excellent  literary  review  of  the  subject,  has  shown  that 
its  use  has  reduced  the  death-rate  from  about  80  to  40  per  cent.,  and 
that  it  therefore  cannot  be  looked  upon  as  a  failure. 

Irons§  has  analyzed  225  cases  of  tetanus  treated  with  antitoxic 
serum  and  found  the  mortality  20  per  cent,  lower  than  in  cases 
otherwise  treated.  He  says  that  it  is  important  that  the  full  effect 
of  the  antitoxin  be  immediately  obtained,  the  best  method  of  using 
it  being  that  outlined  by  Park  in  which  3000  units  are  given  intra- 
spinously  at  the  earliest  possible  moment  after  the  symptoms  ap- 
pear, and  10,000  to  20,000  units  given  intravenously  at  the  same 
time.  On  the  following  day  the  intraspinous  injection  of  3000 
should  be  repeated.  On  the  fourth  or  fifth  day,  10,000  units  should 
be  given  subcutaneously.  By  these  means  a  high  antitoxic  content 
of  the  blood  and  juices  is  maintained. 

The  use  of  antitoxic  serums  must  not  replace  other  non-specific 
modes  of  treatment  such  as  local  treatment  of  the  wound  and  the 
administration  of  sedatives,  etc.  The  result  of  its  experimental  in- 
jection, in  combination  with  the  toxin,  into  mice,  guinea-pigs,  rab- 
bits, and  other  animals  is  perfectly  satisfactory,  and  affords  protec- 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Dec.,  1897,  Bd.  xxn,  Nos.  22,  23,  p.  663. 
'  "Bulletin  of  the  Johns  Hopkins  Hospital,"  July  and  August,  1895. 
j  "Annals  of  Surgery,"  1900,  xxxn,  2,  pp.  219,  416,  567. 
§"Jour.  Am.  Med.  Asso.,"  1914,  LXII,  2025. 


350 


Tetanus 


tion  against  almost  any  multiple  of  the  fatal  dose,  but  the  quantity 
needed,  in  proportion  to  the  body-weight,  to  save  an  animal  from  the 
unknown  quantity  of  toxin  being  manufactured  in  its  body  increases 
so  enormously  with  the  day  or  hour  of  the  disease  as  to  make  the 
dose,  which  increases  millions  of  times  where  that  of  diphtheria  anti- 
toxin increases  but  tenfold,  a  matter  of  difficulty  and  uncertainty. 
Nocard  also  called  attention  to  the  fact  that  the  existence  of  tetanus 
cannot  be  known  until  a  sufficient  toxemia  to  produce  spasms  exists, 
and  that  therefore  it  is  impossible  to  attack  the  disease  in  its  incep- 
tion or  to  begin  the  treatment  until  too  late  to  effect  a  cure.  At  this 
point  it  is  well  to  recall  Nocard's  experiment  with  the  sheep,  in  whose 
blood  so  much  toxin  was  already  present  when  symptoms  first  ap- 
peared that  the  amputation  of  their  infected  tails  could  not  save 
them. 

The  explanation  of  this  inability  of  the  antitoxin  to  effect  a  cure 
,  when  administered  after  development  of  the  symptoms  of  tetanus  is 
probably  found  in  a  ready  fixation  of  the  toxin  in  the  bodies  of  the 
infected  animals.  This  is  well  shown  by  the  experiments  of  Db'nitz,* 
who  found  that  if  a  mixture  of  toxin  and  antitoxin  were  made  before 
injection  into  an  animal,  twelve  minimum  fatal  doses  were  neutralized 
by  i  cc.  of  a  i  :  2000  dilution  of  an  antitoxin.  If,  however,  the 
antitoxin  was  administered  four  minutes  after  the  toxin,  i  cc.  of  a 
i  :  600  dilution  was  required;  if  eight  minutes  after,  i  cc.  of  a  i  :  200 
dilution;  if  fifteen  minutes  after,  i  cc.  of  a  i  :  100  dilution.  He  found 
that  similar  but  slower  fixation  occurred  with  diphtheria  toxin. 

It  was  found  by  Roux  and  Borrelf  that  doses  of  tetanus  antitoxin 
absolutely  powerless  to  affect  the  progress  of  the  disease,  when  ad- 
ministered in  the  ordinary  manner  by  subcutaneous  injection,  read- 
ily saved  the  animal  if  the  antitoxin  were  injected  into  the  brain 
substance. 

Chauffard  and  Quenu,J  who  injected  the  antitoxin  into  the 
cerebral  substance,  found  that  such  administration  brought  about 
an  apparent  cure  in  one  case. 

Their  observations  were  followed  by  an  attempt  to  apply  the 
method  in  human  medicine,  and  patients  with  tetanus  were  trephined 
and  the  antitoxin  injected  beneath  the  dura  and  into  the  cerebral 
substance.  The  results  have  not,  however,  been  satisfactory,  and 
as  the  method  cannot  be  looked  upon  as  itself  free  from  danger,  it 
has  been  abandoned. 

The  only  means  of  treating  the  disease  to  be  recommended  at 
present  is  the  intraspinous,  intravenous  and  subcutaneous  injection 
of  large  and  frequently  repeated  doses  of  the  antitoxic  serum.  There 
can  be  little  doubt  but  that  the  administration  must  be  so  free  as  to 
load  up  the  patient's  blood  with  the  antitoxin  in  hopes  that  its  pres- 

*  Reference  18,  in  "Jour,  of  Hygiene,"  vol.  n,  No.  2,  in  Ritchie's  article. 

"Ann.  de  1'Inst.  Pasteur,"  1898,  No  4 
t  "La  Presse  med.,"  No.  5,  1898 


Bacilli  Resembling  the  Tetanus  Bacillus  351 

ence  there  may  detach  the  toxic  molecules  from  their  anchorage  to 
the  nerve  cells. 

Prophylactic  Treatment. — While  tetanus  antitoxin  is  extremely 
disappointing,  in  practice,  for  the  cure  of  tetanus,  it  is  most  satis- 
factory for  its  prevention.  "  An  ounce  of  prevention  is  better  than  a 
pound  of  cure,"  and  if  the  surgeon  would  administer  a  prophylactic 
injection  of  tetanus  antitoxin  in  every  case  in  which  the  occurrence 
of  tetanus  was  at  all  likely,  the  disease  would  rarely  develop. 

BACILLI  RESEMBLING  THE  TETANUS  BACILLUS 

Tavel*  has  called  attention  to  a  bacillus  commonly  found  in  the  intestine, 
sometimes  in  large  numbers  in  the  appendix  in  cases  of  appendicitis,  and  looked 
upon  by  one  of  his  colleagues,  Fraulein  Dr.  von  Mayer,  as  the  probable  common 
cause  of  appendicitis.  He  calls  it  the  "Pseudo-tetanus-bacillus." 

The  bacillus  measures  0.5  by  5-7 ju,  is  rather  more  slender  than  the  tetanus 
bacillus,  and  its  spores  are  oval,  situated  at  the  end  of  the  rod,  and  cause  a  slight 
bulging  rather  pointed  at  the  end.  The  bacillus  is  provided  with  not  more  than 
a  dozen  flagella — usually  only  four  to  eight — thus  differing  markedly  from  the 
tetanus  bacillus,  which  has  many.  The  flagella  are  easily  stained  by  Loffler's 
method  without  the  addition  of  acid  or  alkali.  The  organism  does  not  stain  so 
well  by  Gram's  method  as  the  true  tetanus  bacillus.  The  bacillus  is  a  pure 
anaerobe. 

The  growth  in  bouillon  is  rather  more  rapid  than  that  of  the  tetanus  bacillus. 
It  will  not  grow  in  gelatin.  The  growth  in  agar-agar  is  very  luxuriant  and 
accompanied  by  the  evolution  of  gas.  Upon  obliquely  solidified  agar-agar  the 
colonies  are  round,  circumscribed,  and  often  encompassed  by  a  narrow,  clear 
zone,  which  is  often  notched.  The  spores  are  killed  at  8o°C. 

The  organism  produced  no  symptoms  in  mice,  guinea-pigs,  and  rabbits  even 
when  2-5  cc.  of  a  culture  were  subcutaneously  introduced. 

Sanfelicef  and  Lubinskit  have  observed  a  bacillus  in  earth  and  meat-infusions 
that  is  morphologically  and  culturally  like  the  tetanus  bacillus,  but  differs  from 
it  in  not  possessing  any  pathogenic  powers. 

Kruse§  has  also  described  a  bacillus  much  like  the  tetanus  micro-organism  that 
grows  aerobically.  It  is  not  pathogenic. 

*  "Centralbl.  f.  Bakt.,"  etc.,  March  31,  1898,  xxm,  No.  13,  p.  538. 

t  "Zeitschrift  fiir  Hygiene,"  vol.  xrv. 

j  "  Centralbl.  f .  Bakt.  u.  Parasitenk.,"  xvi,  19. 

§  Fliigge,  "  Die  Mikroorganismen,"  vol.  n,  p.  267. 


CHAPTER  IV 
ANTHRAX 

BACILLUS  ANTHRACIS  (KOCH) 


General  Characteristics.— A  non-motile,  non-flagellated,  sporogenpus,  liquefy- 
g,  non-chromogenic,   pathogenic,   aerobic  bacillus  staining  by  the  ordinary 


methods  and  by  Gram's  method. 


The  disease  of  herbivora  known  as  anthrax,  "  splenic  fever," 
"Milzbrand"  and  "charbon"  is  a  dreaded  and  common  malady  in 
France,  Germany,  Hungary,  Russia,  Persia,  and  the  East  Indian 
countries.  In  Siberia  the  disease  is  so  common  and  malignant  as  to 


Fig.  127. — Bacillus   anthracis;   colony   three   days   old   upon   a   gelatin   plate 
adhesive  preparation.     X    1000   (Frankel  and  Pfeiffer). 

deserve  its  popular  name,  "  Siberian  pest."  Certain  districts,  as  th< 
Tyrol  and  Auvergne,  in  which  it  seems  to  be  endemic,  serve  as  foe 
from  which  the  disease  spreads  in  summer,  afflicting  many  animals 
and  ceasing  its  depredations  only  with  the  advent  of  winter.  It  i 
not  rare  in  the  United  States,  where  it  seems  to  be  chiefly  a  diseas 
of  the  summer  season. 

Herbivorous  animals  are  most  frequently  affected,  especiall; 
cows  and  sheep.  Carnivorous  animals  are  less  often  affected 
though  not  immune.  Among  laboratory  animals,  white  mice,  house 
mice,  guinea-pigs,  and  rabbits  are  highly  susceptible;  rats,  scarcel; 

352 


Speculation 


353 
Man 


susceptible;  birds,  reptiles  and  amphibians  usually  immune, 
is  susceptible  in  varying  degree. 

Anthrax  was  one  of  the  first  infectious  diseases  proved  to  depend 
upon  a  specific  micro-organism.  As  early  as  1849  Pollender*  dis- 
covered small  rod-shaped  bodies  in  the  blood  of  animals  suffering 
from  anthrax,  but  the  exact  relation  which  they  bore  to  the  disease 
was  not  pointed  out  until  1863,  when  Davaine,fbya  series  of  in- 
teresting experiments,  proved  their  etiologic  significance  to  most 
unbiased  minds.  The  final  confirmation  of  Davaine's  conclusions 
and  actual  proof  of  the  matter  rested  with  Koch,|  who,  observing 


Fig.  128. — Bacillus  anthracis;  showing  the  capsules.     From  a  case  of  human 
infection.     Magnified    1000    diameters    (Schwalbe). 

that  the  bacilli  bore  spores,  cultivated  them  successfully  outside 
the  body,  and  produced  the  disease  by  the  inoculation  of  pure 
cultures. 

Morphology. — The  anthrax  bacillus  is  a  large  rod-shaped  organ- 
ism, of  rectangular  form,  with  slightly  rounded  corners.  It  meas- 
ures 5  to  20  JJL  in  length  and  from  i  to  1.25/4  in  breadth.  It  has  a 
pronounced  tendency  to  form  long  threads,  in  which,  however,  the 
individuals  can  usually  be  made  out,  the  lines  of  junction  of  the  com- 
ponent bacilli  giving  the  thread  somewhat  the  appearance  of  a 
bamboo  rod.  In  preparations  made  by  staining  blood  or  other 
animal  juices  the  bacilli  often  appear  surrounded  by  transparent 
capsules.  Such  are  not  found  in  specimens  made  from  artificial 
cultures. 

Sporulation. — The  formation  of  endospores  is  prolific  in  the  pres- 

*  "Vierteljahrsschr.  fur  ger.,  Med.,"  1855,  Bd.  vm. 
t  "Compte-rendu,"  1863,  Ivii. 
t  "Beitrage  zur  Biol.  d.  Pflanzen,"  1876,  n. 
23 


354  Anthrax 

ence  of  oxygen.  When  oxygen  is  withheld  spore-formation  does 
not  occur.  In  the  bodies  of  experiment  animals  spore-formation 
is  unusual  and  its  occurrence  signifies  the  local  presence  of  abundant 
oxygen.  On  account  of  this  peculiarity  of  the  organism,  the  dead 
body  of  an  animal  is  less  dangerous  as  a  source  of  infection  than  the 
discharges  from  living  animals.  As,  however,  the  wool,  hair  and 
hides  of  infected  animals  are  always  soiled  by  the  discharges,  these 
are  a  menace  to  all  that  handle  them  and  ought  not  be  used.  Each 
spore  has  a  distinct  oval  shape,  is  transparent,  situated  at  the  center 
of  the  bacillus  in  which  it  occurs.  It  does  not  alter  the  contour  of 
the  bacillus.  When  a  spore  is  placed  under  conditions  favorable 
to  its  development,  it  increases  in  length  and  ruptures  at  the  end, 


Fig.  129. — Bacillus  anthracis,  stained  to  show  the  spores.     X    1000  (Frankel 

and  Pfeiffer). 

from  which  the  new  bacillus  escapes.  The  spores  of  the  anthrax 
bacillus,  being  large  and  readily  obtainable,  form  excellent  subjects 
for  the  study  of  spore-formation  and  germination,  for  the  study  of 
the  action  of  germicides  and  antiseptics,  and  for  staining. 

Motility.— The  bacilli  are  not  motile  and  have  no  flagella. 

Staining.— They  stain  well  with  ordinary  solutions  of  the  anilin 
dyes,  and  can  be  beautifully  demonstrated  in  the  tissues  by  Gram's 
method  and  by  Weigert's  modification  of  it.  Picrocarmin,  followed 
by  Gram's  stain,  gives  a  beautiful,  clear  picture.  The  spores  can 
be  stained  by  any  of  the  special  methods  (q.v.). 

Isolation.— The  bacillus  of  anthrax  is  one  of  the  easiest  organisms 
to  secure  in  pure  culture  from  the  tissues  and  excreta  of  diseased 
animals.  Its  luxurious  vegetation,  the  typical  appearance  of  its 
colonies,  and  its  infectivity  for  the  laboratory  animals  combine  to 
make  possible  its  isolation  either  by  direct  cultivation  from  the  tis- 


Cultivation 

sues,  by  the  plate  method,  or  by  the  inoculation  into  animals  and 
recovery  of  the  micro-organisms  from  their  blood. 

Cultivation. — Colonies. — Upon  the  surface  of  a  gelatin  plate  the 
bacillus  forms  beautiful  and  highly  characteristic  colonies.  To  the 
naked  eye  they  appear  first  as  minute  round,  grayish-white  dots. 
Under  the  microscope  they  are  egg-shaped,  slightly  brown  and  granu- 
lar. Upon  the  surface  of  the  medium,  they  spread  out  into  flat, 
irregular,  transparent  tufts  like  curled  wool,  and  from  a  tangled  cen- 
ter large  numbers  of  curls,  made  up  of  parallel  threads  of  bacilli, 
extend  upon  the  gelatin.  Before  the  colony  attains  to  any  consider- 
able size  liquefaction  sets  in.  Beautiful  adhesion  preparations  can 


Fig.  130. — Bacillus  anthracis;  colony  upon  a  gelatin  plate.     X    100  (Frankel 

and    Pfeiffer). 

be  made  if  a  perfectly  clean  cover-glass  be  passed  once  through  a 
flame  and  laid  carefully  upon  the  gelatin,  the  colonies  being  picked 
up  entire  as  the  glass  is  carefully  removed.  Such  a  specimen  can 
be  dried,  fixed,  and  stained  in  the  same  manner  as  an  ordinary  cover- 
glass  preparation. 

Gelatin  Punctures. — In  gelatin  puncture  cultures  the  growth  is 
even  more  characteristic  than  are  the  colonies.  The  bacilli  begin  to 
grow  along  the  entire  track  of  the  wire,  but  develop  most  luxuriantly 
at  the  surface,  where  oxygen  is  plentiful  and  where  a  distinct  shaggy 
pellicle  is  formed.  From  the  deeper  growth,  fine  filaments  extend 
from  the  puncture  into  the  surrounding  gelatin,  with  a  beautiful 
arborescent  effect. 

Liquefaction  progresses  from  above  downward  until  ultimately 
the  entire  gelatin  is  fluid  and  the  growth  sediments. 

Agar-agar. — Upon  agar-agar  characteristic  appearances  are  few. 
The  growth  takes  place  along  the  line  of  inoculation,  forming  a 


356 


Anthrax 


grayish-white,  translucent,  slightly  wrinkled  layer  with  irregular 
edges,  from  which  curls  of  bacillary  threads 
extend  upon  the  medium.  When  the 
culture  is  old,  the  agar-agar  usually  be- 
comes brown  in  color.  Spore-formation 
is  luxuriant. 

Bouillon. — In  bouillon  the  anthrax  bacil- 
lus grows  chiefly  upon  the  surface,  where 
a  thick  felt-like  pellicle  forms.  From  this, 
fuzzy  extensions  descend  into  the  clear 
bouillon  below.  After  a  few  days  some 
wooly  aggregations  can  be  seen  in  the 
bottom  of  the  tube.  In  the  course  of  time 
the  growth  ceases  and  the  surface  pellicle 
sinks.  If,  by  shaking,  it  is  caused  to  sink 
prematurely,  a  new,  similar  surface  growth 
takes  its  place.  Spore-formation  is  rapid 
at  the  surface. 

Potato.— Upon  the  potato  the  growth  is 
white,  creamy,  and  rather  dry.  Sporula- 
tion  is  marked. 

Blood-serum. — Blood-serum  cultures 
lack  characteristic  peculiarities;  the  cul- 
ture-medium is  slowly  liquefied. 

Milk. — The  anthrax  bacillus  grows  well 
in  milk,  which  it  coagulates  and  acidulates. 
Later  the  coagulum  is  peptonized  arid  dis- 
solved, leaving  a  clear  whey. 

Vital  Resistance. — The  bacillus  grows 
between  the  extremes  of  12°  and  45°C., 
best  at  37°C.  The  exposure  of  the  organ- 
ism to  the  temperature  of  42°  to  43°C. 
slowly  diminishes  its  virulence. 

When  dried  upon  threads,  the  spores 
retain  their  vitality  for  years,  and  are 
highly  resistant  to  heat  and  disinfectants. 
The  spores  of  anthrax  are  killed  by  five 
minutes'  exposure  to  ioo°C.  It  is  said 
by  some  that  spores  subjected  to  5  per 
cent,  carbolic  acid  can  subsequently 
germinate  when  introduced  into  suscepti- 
ble animals,  their  resistance  to  this 
strength  carbolic  solution  being  so  great 
that  they  are  not  destroyed  by  it  under 
twenty-four  hours.  They  are  killed  in 

two  hours  by  exposure  to  i  :  1000  bichlorid  of  mercury  solution. 
Metabolic  Products.— The  anthrax  bacillus  produces  a  curdling 


Fig.  131. — Bacillus  an- 
thracis;  gelatin  stab  cul- 
ture, showing  character- 
istic growth  with  com- 
mencing liquefaction  and 
cupping  (from  evapora- 
tion) at  the  surface  of  the 
medium  (Curtis). 


Pathogenesis 


357 


ferment.  Iwanow*  found  that  the  organism  forms  acetic,  formic, 
and  caproic  acids,  but  it  produces  no  important  change  of  reaction  in 
the  medium  in  which  it  grows.  It  gen-, 
crates  no  indol.  Its  proteolytic  enzyme 
is  active,  digesting  both  casein  and  fibrin. 

It  is  doubtful  whether  the  anthrax  bacil- 
lus produces  any  important  toxic  sub- 
stance. Hoffaf  isolated  a  basic  substance 
from  anthrax  cultures  and  called  it  an- 
thracin;  Hankin  and  Wesbrook,{  an  albu- 
mose  fatal  in  large  doses  and  immunizing 
in  small  ones.  Brieger  and  Frankel§ 
isolated  a  tox-albumin  from  the  tissues  of 
animals  dead  of  anthrax.  Martin ||  sepa- 
rated protalbumose,  deuteroalbumose, 
peptone,  an  alkaloid,  leucin,  and  tyrosin. 
The  albumoses  were  not  very  poisonous, 
but  the  alkaloid  was  capable  of  producing 
death  after  the  development  of  somnol- 
ence. The  animals  were  edematous. 
Marmier**  isolated  a  toxin  of  non-albu- 
minous nature  and  immunizing  power. 
Conradift  in  an  elaborate  research  failed 
to  find  that  the  anthrax  bacillus  produced 
any  soluble  extracellular  or  intracellular 
poison  capable  of  affecting  susceptible 
animals,  and  concludes  that  it  is  highly 
improbable  that  the  anthrax  bacillus 
produces  any  toxic  substances  at  all. 

Pathogenesis. — Avenues  of  Infection. — 
Infection  usually  takes  place  through  the 
respiratory  tract,  through  the  alimentary 
canal,  or  through  the  skin.  It  may  take 
place  through  the  placenta. 

I.  The  Respiratory  Tract. — The  inhala- 
tion of  the  spores  of  the  anthrax  bacillus 
is  possible  whenever  such  are  present  in 
the  atmosphere.  The  effect  produced  will 
depend  upon  the  number  of  spores  inhaled 
and  the  resistance  or  susceptibility  of  the 
animal.  In  man,  a  resisting  animal,  an- 


Fig.  132. — Bacillus  an- 
thracis;  glycerin  agar-agar 
culture  (Curtis). 


'Ann.  de  PInst.  Pasteur,"  1892. 

'Ueber  die  Natur.  des  Milzbrandgifts,"  Wiesbaden,  1886. 
'Ann.  de  1'Inst.  Pasteur,"  1892,  No.  9. 
'Ueber  Ptomaine,"  Berlin,  1885-1886. 
'Proceedings  of  the  Royal  Society,"  May  22,  1890. 
"Ann.  de  PInst.  Pasteur,"  1895,  p.  533. 
ft  " Zeitschrif t  fur  Hygiene,"  June  14,  1899. 


358  Anthrax 

thrax  is  rarely  so  caused  except  the  number  of  bacilli  be  great, 
when  it  results  in  a  disturbance  at  first  localized  in  the  lungs,  and 
much  resembling  pneumonia.  From  the  lungs  generalized  infection 
may  later  occur  and  destroy  life.  This  form  of  infection  is  of  occa- 
sional occurrence  among  men  whose  occupation  occasionally  brings 
them  into  contact  with  the  hair  or  hides  of  animals  dead  of  anthrax, 
and  is  often  spoken  of  as  "wool-sorters'  disease." 

Anthrax  in  cattle  probably  results  from  the  inhalation  or  ingestion 
of  the  spores  of  the  bacilli  from  the  pasture.  Interesting  discussions 
arose  concerning  the  infection  of  the  pastures.  It  was  argued  that, 
the  bacilli  being  inclosed  in  the  tissues  of  the  diseased  animals,  in- 
fection of  the  pasture  must  depend  upon  the  distribution  of  the  germs 
from  buried  cadavers,  either  through  the  activity  of  earthworms, 
which  ate  of  the  earth  surrounding  the  corpse  and  deposited  the 
spores  in  their  excrement  (Pasteur),  or  to  currents  of  moisture  in  the 
soil.  Koch  seems,  however,  to  have  demonstrated  the  fallacy  of 
both  theories  by  showing  that  the  conditions  under  which  the  bacilli 
find  themselves  in  buried  cadavers  are  opposed  to  fructification  or 
sporulation,  and  that  in  all  probability  the  bacteria  suffer  the  same 
fate  as  the  cells  of  the  buried  animals,  and  disintegrate,  especially 
if  the  animal  be  buried  at  a  depth  of  two  or  three  meters. 

Frankel  points  out  particularly  that  no  infection  of  the  soil  by 
the  dead  animal  could  be  worse  than  the  pollution  of  its  surface  by 
the  bloody  stools  and  urine,  rich  in  bacilli,  discharged  upon  it  by  the 
animal  before  death,  and  that  it  is  the  live,  and  not  the  dead,  animals 
that  are  to  be  blamed  for  the  infection. 


Fig-  133- — Anthrax  carbuncle  or  malignant  carbuncle  (Lexer). 

II.  The  Alimentary  Tract. — When  the  bacilli  are  taken  into  the 
stomach  they  are  probably  destroyed  by  the  acid  gastric  juice. 
The  spores,  however,  are  able  to  endure  the  acid,  and  pass  uninjured 
into  the  intestine,  where  the  suitable  alkalinity  enables  them  to 
develop  into  bacilli,  surround  the  villi  with  thick  networks  of  bacil- 
lary  threads,  separate  the  covering  epithelial  cells,  enter  the  lym- 
phatics, and  then  the  blood,  and  effect  general  infection. 

///.  The  Skin. — The  bacillus  frequently  enters  the  body  through 
wounds",  cuts,  scratches,  and  perhaps  occasionally  fly-bites,  though 


Pathogenesis 


359 


from  the  work  of  Nuttall*  it  is  pretty  clear  that  flies  play  little  part 
in  the  transmission  of  the  disease.  Under  these  conditions  the  organ- 
isms at  once  find  themselves  in  the  lymphatics  or  capillaries,  and 
may  cause  immediate  general  infection.  In  human  beings  a  "  malig- 
nant pustule"  is  apt  to  follow  local  infection,  and  may  recover  or 
ultimately  cause  death  by  general  infection. 

The  malignant  pustule  usually  makes  its  appearance  upon  the  face, 
hands  or  arms.  The  first  symptom  is  a  reddish  papule  that  extends 
and  becomes  vesicular.  At  the  point  of  infection  necrosis  is  rapid, 
and  within  forty-eight  hours  there  may  be  a  brownish  eschar  sur- 
rounded by  a  crop  of  secondary  vesicles,  beyond  which  there  is  edema 


Fig.  134.— Anthrax  bacilli  in  glomeruli  of  kidney. 

or  brawny  swelling.  According  to  the  susceptibility  of  the  patient 
the  disease  may  soon  localize,  the  slough  detach  and  recovery  set  in, 
or  the  edema  and  swelling  may  continue,  blood  invasion  occur  and 
death  ensue.  Heinemann,f  in  compiling  statistics  of  the  fatality  of 
malignant  pustule,  shows  that  the  danger  of  the  lesion  is  greatly  miti- 
gated by  complete  excision.  Koch  found  the  death-rate  among  1473 
cases  to  be  38.8  per  cent.,  but  Heinemann's  statistics  upon  2255 
cases  show  the  deaths  to  be  only  5.8  per  cent. 

Lesions. — The  disease  as  seen  in  the  laboratory  is  accompanied 
by  few  marked  lesions.  The  ordinary  experimental  inoculation  is 
made  by  cutting  away  a  little  of  the  hair  from  the  abdomen  of  a 
guinea-pig  or  rabbit,  or  at  the  root  of  a  mouse's  tail,  making  a  little 

*  "Johns  Hopkins  Hospital  Reports,"  1899. 
t'-'Deutsche  Zeitschrift  fur  Chirurgie,"  1912,  cxix,  201. 


360  Anthrax 

subcutaneous  pocket  by  a  snip  with  sterile  scissors,  and  introducing 
the  spores  or  bacilli  with  a  heavy  platinum  wire,  the  end  of  which  is 
flattened,  pointed,  and  perforated.  An  animal  inoculated  in  this 
way  dies,  according  to  the  species,  in  from  twenty-four  hours  to 
three  days,  suffering  from  weakness,  fever,  loss  of  appetite,  and  a 
bloody  discharge  from  nose  and  bowels.  There  is  much  subcutane- 
ous edema  near  the  inoculation  wound.  The  abdominal  viscera 
are  injected  and  congested.  The  spleen  is  enlarged,  dark  in  color, 
and  of  mushy  consistence.  The  liver  is  also  somewhat  enlarged. 
The  lungs  are  usually  slightly  congested. 

When  organs  which  present  no  appreciable  changes  to  the  naked 
eye  are  subjected  to  a  microscopic  examination,  the  appropriate 
staining  methods  show  the  capillary  and  lymphatic  systems  to  be 
almost  universally  occupied  by  bacilli,  which  extend  throughout 
their  meshworks  in  long  threads.  Most  beautiful  bacillary  threads 
can  be  found  in  the  glomeruli  of  the  kidney  and  in  the  minute  capil- 
laries of  the  intestinal  villi.  In  the  larger  vessels,  where  the  blood- 
stream is  rapid,  no  opportunity  is  afforded  for  the  formation  of  the 
threads,  and  the  bacteria  are  relatively  few,  so  that  the  burden  of 
bacillary  obstruction  is  borne  by  the  minute  vessels.  The  condition 
is  thus  a  pure  bacteremia. 

Death  from  anthrax  seems  to  depend  more  upon  the  obstruction 
of  the  circulation  by  the  multitudes  of  bacilli  in  the  capillaries,  and 
upon  the  appropriation  of  the  oxygen  destined  to  support  the  tissues, 
by  the  bacilli,  than  upon  intoxication  by  the  metabolic  products 
of  bacillary  growth. 

Virulence. — The  anthrax  bacillus  maintains  its  virulence  almost 
without  modification  because  of  the  prolific  formation  of  spores  and 
their  remarkable  resisting  powers.  By  artificial  means,  however, 
the  formation  of  spores  can  be  inhibited  and  the  bacilli  attenuated. 
This  was  first  achieved  by  Pasteur*  by  cultivation  at  temperatures 
above  the  optimum,  at  which  no  spores  were  formed.  Toussaintf 
found  that  the  addition  of  i  per  cent,  of  carbolic  acid  to  blood  of 
animals  dead  of  anthrax  destroyed  the  virulence  of  the  bacilli; 
ChamberlandJ  and  Roux  found  the  virulence  destroyed  when  0.1-0.2 
per  cent,  of  bichromate  of  potassium  was  added  to  the  culture 
medium;  Chauveau  used  atmospheric  pressure  to  the  extent  of  six 
to  eight  atmospheres  and  found  the  virulence  diminished;  Arloing§ 
found  that  direct  sunlight  operated  similarly;  Lubarsch,  that  the 
inoculation  of  the  bacilli  into  immune  animals,  such  as  the  frog, 
and  their  subsequent  recovery  from  its  blood,  diminishes  the 
virulence. 

Vaccination. — Pasteurll  early  realized  the  importance  of  some  prac- 

*"Rec.  de  med  vet.,"  Paris,  1879,  P-  193. 
'  "  Compte-rendu  Acad.  des  Sci.  de  Paris,"  xci,  1880,  p.  135. 
"Ann.  de  1'Inst.  Pasteur,"  1894,  p.  161. 

§  "Compte-rendu  de  1'Acad.  des  Sci.,"  Paris,  1892,  cxiv,  p.  1521. 
II  "Rec.  de  MeU  vet.,"  Paris,  1879,  p.  193. 


Bacteriologic  Diagnosis  301 

tical  measure  for  the  protective  vaccination  of  cattle  against  the 
disease,  and  devoted  himself  to  investigating  the  problem.  He 
found  that  the  inoculation  of  attenuated  bacilli  into  cows  and  sheep, 
and  their  subsequent  reinoculation  with  mildly  virulent  bacilli, 
afforded  them  immunity  against  highly  virulent  organisms. 

The  protective  inoculations  prepared  by  Pasteur  consisted  of 
two  cultures  of  diminished  virulence,  to  be  employed  one  after  the 
other,  each  rendering  the  vaccinated  animals  more  immune.  The 
cultures  were  prepared,  that  is,  attenuated  by  cultivation  at  42° C. 
for  a  sufficient  length  of  time,  the  bacilli  forming  no  spores  and 
gradually  losing  their  virulence  at  this  temperature.  The  first 
vaccine  was  kept  from  fifteen  to  twenty  days  at  42°C.  It  killed 
mice  and  guinea-pigs  one  day  old,  but  was  without  action  on  guinea- 
pigs  of  adult  size.  The  second  vaccine  only  remained  at  the  tem- 
perature of  42°C.  for  from  ten  to  twelve  days  and  killed  mice, 
guinea-pigs  and  occasionally  rabbits. 

The  second  vaccine  is  administered  from  two  to  three  weeks  after 
the  first  is  given,  by  hypodermic  injection  into  the  tissues  of  the  neck 
or  flank.  Of  each  broth  culture  about  i  cc.  is  administered.  The 
animals  frequently  become  ill. 

Pasteur  demonstrated  the  value  of  his  method  in  1881  at  Pouilly- 
le-Fort,  in  a  manner  so  convincing  to  the  entire  world  that  it  was 
immediately  put  into  practice  in  France.  Roger*  says  that  between 
1882  and  1894  there  were  1,788,677  sheep  vaccinated,  with  a  mor- 
tality of  0.94  per  cent.,  the  previous  death-rate  having  been  10  per 
cent.  There  were  also  200,962  cattle  vaccinated,  with  a  reduction 
of  the  death-rate  from  5  per  cent,  to  0.34  per  cent. 

Slight  protection  against  anthrax  can  be  afforded  in  other  ways. 
Hiippe  found  that  the  simultaneous  inoculation  of  bacteria  not  at  all 
related  to  anthrax  will  sometimes  cause  the  animal  to  recover.  Han- 
kin  found  in  the  cultures  chemic  substances,  especially  an  albuminose, 
that  exerted  a  protective  influence.  Rettgerf  prepared  "  prodigiosus 
powder"  from  potato  cultures  of  B.  prodigiosus,  which  when  in- 
jected into  guinea-pigs  during  experimental  anthrax  infection  pro- 
longed life  or  induced  recovery. 

Serum  Therapy. — In  1890  Ogata  and  Jasuhara  showed  that  the 
blood  of  experiment  animals  convalescent  from  anthrax  possessed 
an  antitoxic  substance  of  such  strength  that  i :  800  parts  per  body- 
weight  would  protect  a  mouse.  Similar  results  have  been  attained 
by  Marchoux.J  Serum  therapy  in  anthrax  is,  however,  of  no  prac- 
tical importance  either  for  prophylaxis  or  treatment,  as  vaccinating 
the  animals  is  far  cheaper  and  more  satisfactory. 

Bacteriologic  Diagnosis. — When  it  is  desired  to  have  a  bacterio- 
logic  diagnosis  of  anthrax  made  where  no  laboratory  facilities  are  at 

*Les  Maladies  Infectieuse,  n,  p.  1489. 

t  "Jour,  of  Infectious  Diseases,"  Nov.  25,  1905,  vol.  n,  No.  4,  p.  S62- 

I  "Ann.  de  1'Inst.  Pasteur,"  November,  1895,  ix,  No.  n,  pp.  5°-75- 


362  Anthrax 

hand,  an  ear  of  the  dead  animal  can  be  inclosed  in  a  bottle  or  frui 
jar  and  sent  to  the  nearest  laboratory  where  diagnosis  can  be  made 
The  ear  contains  so  little  readily  decomposable  tissue  that  it  keep: 
fairly  well,  drying  rather  than  rotting.  It  contains  enough  blood  t( 
enable  a  bacteriologist  to  make  a  successful  examination. 

Sanitation. — As  every  animal  affected  with  anthrax  is  a  menace  tc 
the  community  in  which  it  lives — to  the  men  who  handle  it  as  wel 
as  the  animals  who  browse  beside  it — such  animals  should  be  killec 
as  soon  as  the  diagnosis  is  made,  and,  together  with  the  hair  and  skin 
be  burned,  or  if  this  be  impracticable,  Frankel  recommends  that  the} 
be  buried  to  a  depth  of  at  least  i  J^-2  meters,  so  that  the  sporulatior 
of  the  bacilli  is  made  impossible.  The  dejecta  should  also  be  care 
fully  disinfected  with  5  per  cent,  carbolic  acid  solution.  As  th< 
pastures  and  barnyards  are  certainly  infected  wherever  an  anima 
has  been  the  victim  of  anthrax,  all  other  susceptible  animals  upor 
the  farm,  and  all  such  upon  neighboring  farms,  should  at  once  b< 
vaccinated. 

Cases  of  human  anthrax  must  be  treated  by  isolation,  carefu 
dressing  of  the  lesions  when  external,  the  dressings  being  burnec 
as  soon  as  removed.  The  expectoration,  urine  and  feces  should  b< 
disinfected  with  care.  The  patient  should  be  defended  from  flies 
and  the  nurse  and  others  who  come  into  contact  with  the  patiem 
should  be  warned  of  the  dangerous  character  of  the  infection. 

BACILLI  RESEMBLING  THE  ANTHRAX  BACILLUS 

Bacilli  presenting  the  morphologic  and  cultural  characteristics 
of  the  anthrax  bacillus,  but  devoid  of  any  disease-producing  power 
are  occasionally  observed.  Of  these,  Bacillus  anthracoides  of  Hupp* 
and  Wood,*  Bacillus  anthracis  similis  of  McFarland,f  and  Bacillus 
pseudoanthracisj  have  been  given  special  names.  What  relation- 
ship they  bear  to  the  anthrax  bacillus  is  uncertain.  They  may  be 
entirely  different  organisms,  or  they  may  be  individuals  whose  viru- 
lence has  been  lost  through  unfavorable  environment. 

*  "Berliner  klin.  Wochenschrift,"  1889.  16. 
'  "Centralbl.  f.  Bakt.,"  vol.  xxiv,  No.  26,  p.  556. 
I  "Hygienische  Rundschau,"  1894,  No.  8. 


CHAPTER  V 
HYDROPHOBIA,  LYSSA,  OR  RABIES 

NEURORRHYCTES  HYDROPHOBIA  (CALKINS) 

HYDROPHOBIA,  lyssa,  or  rabies  is  a  specific  infectious  toxic  disease 
to  which  dogs,  wolves,  skunks  and  cats  are  highly  susceptible,  and 
which,  through  their  saliva,  can  be  communicated  to  men,  horses, 
cows  and  other  animals.  The  means  of  communication  is  almost 
invariably  a1>ite,  hence  the  specific  infection  must  be  present  in  the 
saliva. 

The  infected  animals  manifest  no  symptoms  during  a  varying  in- 
cubation period  in  which  the  wound  heals  kindly.  For  human  be- 
ings this  period  may  be  of  twelve  months' duration;  in  rare  cases 
may  be  only  a  few  days;  its  average  duration  is  about  six  weeks. 

Toward  the  close  of  the  incubation  period  an  observable  alteration 
occurs  in  the  wound,  which  becomes  reddened,  may  suppurate,  and 
is  painful.  The  victim  has  a  sensation  of  horrible  dread,  which 
passes  into  wild  excitement,  with  paralysis  of  the  pharyngeal  mus- 
cles and  inability  to  swallow.  The  wild  delirium  ends  in  a  final  stage 
of  convulsion  or  palsy.  The  convulsions  are  tonic,  rarely  clonic,  and 
finally  cause  death  by  interfering  with  respiration. 

During  the  convulsive  period  much  difficulty  is  experienced  in 
swallowing  liquids,  and  it  is  supposed  that  the  popular  term  "hydro- 
phobia" arose  from  the  reluctance  of  the  diseased  to  take  water  be- 
cause of  painful  spasms  brought  on  by  the  attempt. 

The  infectious  nature  of  rabies  seems  to  have  been  first  demon- 
strated by  Gal  tier.*  Pasteur,  Chamberland  and  Rouxf  continued 
the  investigation  and  found  that  in  animals  that  die  of  rabies  the 
salivary  glands,  pancreas  and  the  nervous  system  contain  the 
infection,  and  are  more  appropriate  for  the  experimental  purposes 
than  the  saliva,  which  is  invariably  contaminated  with  accidental 
pathogenic  bacteria. 

The  introduction  of  a  fragment  of  the  medulla  oblongata  of  a  dog 
dead  of  rabies  beneath  the  dura  mater  of  a  rabbit  causes  the  de- 
velopment of  typical  rabies  in  the  rabbit  in  about  six  days. 

Specific  Organism. — It  is  not  yet  generally  conceded  that  the 
pathogenic  micro-organism  of  rabies  has  been  discovered,  though  there 
is  continually  accumulating  evidence  in  favor  of  the  "bodies  of 
Negri."J  Believing  that  the  evidence  at  hand  is  strongly  in  favor 

*  "Compte-rendu  de  1'Acad.  des  Sciences  de  Paris,"  1879,  LXXXIX,  444. 

flbid.,  i88i,xcn,  159. 

{"Zeitschrift  fur  Hygiene,"  1903,  XLIII,  507;  XLIV,  520;  1909,  LXII,  421 

363 


364  Hydrophobia,  Lyssa,  or  Rabies 

of  the  protozoan  nature  and  etiological  importance  of  these  bodies, 
they  are  tentatively  accepted  as  the  cause  of  the  disease  and  treated 
accordingly  in  the  text.  To  these  bodies  Calkins  has  given  the 
name  Neurorrhyctes'  hydrophobias. 

Morphology. — By  appropriately  staining  sections  of  the  cerebrum, 
cerebellum,  pons,  basal  ganglia,  spinal  ganglia,  and  salivary  glands, 
of  human  beings  or  animals  dead  of  rabies,  it  was  possible  to  demon- 
strate small  rounded  bodies  measuring  4  to  lo.p.  as  a  rule,  though 
varying  from  i  to  20  /z  in  the  interior  of  the  protoplasmic  process  of 
the  cells.  In  experimental  infections  they  are  most  numerous  in 
the  hippocampal  convolution.  The  bodies,  when  stained  by  the 
methods  given  below,  usually  appear  red  in  color.  They  are  ovoid 
in  shape,  well-circumscribed,  and  vary  in  size  from  invisibility  to 
20  fji  in  length.  The  smaller  of  them  do  not  show  any  structural 
differentiation,  but  the  larger  show  central  condensations  that  may 
be  nuclear  material.  The  greater  number  of  them  lie  in  the  cyto- 
plasm of  the  nerve  cells;  some  are  free.  These  are  the  Negri  bodies. 

Williams  and  Lowden*  are  convinced  that  they  are  protozoan 
organisms,  that  they  are  the  cause  of  rabies,  and  that  their  presence 
is  pathognomonic  of  rabies.  They  believe: 

1.  The  smear  method  of  examining  the  Negri  bodies  (vide  infra)  is  superior 
to  any  other  method  so  far  published  for  the  following  reasons :  (a)  It  is  simpler, 
shorter  and  less  expensive;  (6)  the  Negri  bodies  appear  much  more  distinct 
and  characteristic.     For  this  reason  and  the  preceding  one  its  value  in  diagnostic 
work  is  great;  (c)  the  minute  structure  of  the  Negri  bodies  can  be  demonstrated 
more  clearly;  (d)  characteristic  staining  reactions  are  brought  out. 

2.  The  Negri  bodies  as  shown  by  the  smears,  as  well  as  by  the  sections,  are 
specific  to  hydrophobia. 

3.  Numerous  "bodies"  are  found  in  fixed  virus. 

4.  "Bodies"  are  found  before  the  beginning  of  visible  symptoms,  i.e.,  on  the 
fourth  day  in  fixed  virus,  on  the  seventh  day  in  street  virus,  and  evidence  is  given 
that  they  may  be  found  early  enough  to  account  for  the  appearance  of  infectivity 
of  the  host  tissues. 

5.  Forms  similar  in  structure  and  staining  qualities  to  the  others,  but  just 
within  the  limits  of  visible  structure  (at  1500  diameter  magnification),  have  been 
seen;  such  tiny  forms,  considering  the  evidence  they  give  of  plasticity,  might  be 
able  to  pass  the  coarser  Berkefeld  filters. 

6.  The  Negri  bodies  are  organisms  belonging  to  the  class  Protozoa.     The 
reasons  for  this  conclusion  are:  (a)  They  have  a  definite  characteristic  morphology; 
(b)  this  morphology  is  constantly  cyclic,  i.e.,  certain  forms  always  preponderate 
in  certain  stages  of  the  disease,  and  a  definite  series  of  forms  indicating  growth 
and  multiplication  can  be  demonstrated;  (c)  the  structure  and  staining  quali- 
ties, as  shown  especially  by  the  smear  method  of  examination,  resemble  those 
of   certain   known   Protozoa,   notably   of   those    belonging    to    the   sub-order 
Microsporidia. 

7.  The  proof  that  the  Negri  bodies  are  living  organisms  is  sufficient  proof 
that  they  are  the  cause  of  hydrophobia;  a  single  variety  of  living  organisms  found 
in  such  large  numbers  in  every  case  of  a  disease,  and  only  in  that  disease,  appear- 
ing at  the  time  that  the  host  tissue  becomes  infective,  in  regions  that  are  infect- 
ive, and  increasing  in  those  infective  areas  with  the  course  of  the  disease  can 
be  no  other,  according  to  our  present  views,  than  the  cause  of  that  disease. 

One  of  the  objections  urged  against  the  bodies  of  Negri  as  the 
specific  cause  of  the  disease  was  the  failure  of  the  organism 

*"Jour.  of  Infectious  Diseases,"  1906,  m,  452. 


3   , 


PLATE   1 


* 


^  » 


• 


\. 


• 


Nerve-cells  containing  -Negri  bodies.  Hippocampus  impression  preparation, 
dog.  Van  Gieson  stain;  X  1000.  i,  Negri  bodies;  2,  capillary;  3,  free  red 
blood-corpuscles.  (Courtesy  of  Langdon  Frothingham.) 


Morphology 


365 


to  appear  elsewhere  than  in  the  central  nervous  system,  when  the 
saliva,  the  salivary  glands  and  the  pancreas  were  known  to  harbor 
it.  This  has  now  been  overcome  by  the  demonstration  of  the  bodies 
in  the  salivary  glands  in  precisely  the  same  form  as  that  seen  in  the 
nervous  system  by  Manuelian.* 

Steinhardt,  Poor  and  Lambertf  have  endeavored  to  determine 


whether  Negri  bodies  are  parasitic  micro-organisms  or  degeneration 
products  of  the  nervous  system,  and  have  shown  that  when  cells 
of  the  normal  guinea-pig  brain  are  incubated  in  blood  plasma,  their 
cytoplasm,  when  stained  by  Van  Gieson's  stain,  show  small  pink- 
staining  bodies  surrounded  by  a  blue  granular  ring,  indistinguishable 

*"Ann.  del'Inst.  Pasteur,"  1914,  xxvn,  233. 
f  Jour,  of  Infectious  Diseases,  1912,  xi,  459. 


366 


Hydrophobia,  Lyssa,  or  Rabies 


from  the  unstructured  Negri  bodies  observed  with  great  frequency 
in  the  rabid  guinea-pig  brain.  In  a  few  instances  these  forms  con- 
tained a  blue-staining  central  ring  or  point,  and  closely  resembled 
the  structured  forms  of  Negri  bodies.  The  normal  guinea-pig  brain 
inoculated  with  rabid  material,  street  or  fixed  virus,  incubated  in  the 
same  manner,  showed  the  same  structures.  The  brains  of  guinea- 
pigs  dying  of  street  virus  and  rabbits  dying  of  fixed  virus,  incubated 
in  small  fragments,  gave  no  development  of  Negri  bodies  in  blood 
plasma,  beyond  the  small  structured  and  unstructured  forms,  al- 
though in  one  preparation  the  ganglion  cells  appeared  to  be  living 
at  the  end  of  twenty-one  days'  incubation. 

Cultivation. — Attempts  to  cultivate  Negri  bodies  were  made  by 
Moon,*  but  the  success  of  his  attempts  seemed  doubtful.     The  first 


AO 
BO 


Fig.  136.— From  rabbit  "fixed- virus"  brain;  a,  b,  c,  d,f,  and  j,  types  of  Negri 
bodies  seen  at  death  of  rabbit;  e,g,h,  and./,  apparent  multiplication  and  segmen- 
tation of  the  bodies  after  three  days  at  24°C.  Drawing  made  from  smears 
stained  by  Giemsa's  method  and  magnified  about  2000  diameters  (Williams,  in 
Jour.  Am.  Med.  Assoc.). 

claim  to  successful  cultivation  of  the  Negri  bodies  was  made  by 
Noguchi.f  The  cultivation  was  done  according  to  his  already  suc- 
cessful method  for  Spirochaeta  of  various  kinds.  Large,  small  and 
dividing  bodies  appeared  in  the  culture  fluid,  after  inoculation  with  a 
fragment  of  nervous  tissue  from  various  animals  with  infection  fol- 
lowing inoculation  with  street  virus  and  "fixed"  virus.  But  Wil- 
liams $  at  once  pointed  out  that  there  is  no  certainty  that  the  bodies 
increased  in  numbers  in  the  cultures,  though  Noguchi  says  that  they 
reappear  in  new  cultures  "through  many  generations."  Noguchi's 

*  "Jour,  of  Infectious  Diseases,"  1913,  xm,  213. 

f    Jour,  of  Experimental  Medicine,"  1913,  xvm,  314. 

t"Jour.  Amer.  Med.  Assoc.,"  1913,  LXI,  1509. 


Staining  367 

paper  seems  more  like  a  preliminary  report  than  a  finished  work,  and 
future  publication  on  the  subject  is  promised.  Two  methods  of 
obtaining  the  virus  of  rabies  freed  from  the  cells  of  the  host  and  free 
from  contaminating  organisms,  published  by  Poor  and  Steinhardt,* 
give  some  promise  of  permitting  the  introduction  of  the  bodies  of 
rabies  into  artificial  culture  media  in  a  measured  quantity  of  fluid, 
perhaps  containing  a  known  number  of  organisms,  and  thus  permit- 
ting better  methods  of  estimating  the  growth  in  artificial  culture. 


id  2. 


* 


H 


Fig.  137. — From  dog  "  street- virus "  brain;  a,  b,  c,  and  /,  types  of  Negri 
bodies  seen  at  death  of  dog;  d,  e,  g,  and  A,  apparent  multiplication  and  segmenta- 
tion of  the  bodies  after  three  days  at  24°C.  (Williams,  in  Jour.  Am.  Med.  Assoc.). 


Staining. — The  Negri  bodies  are  not  difficult  to  stain  and  find 
when  one  is  familiar  with  them  or  when  they  are  present  in  the 
nervous  tissue  in  considerable  numbers.  To  find  a  few,  to  find  them 
quickly,  and  to  recognize  them  unmistakably  is,  however,  a  different 
matter.  They  stain  by  all  of  the  Romanowsky  modifications,  by  all 
of  the  eosin-methylene  blue  combinations,  and  by  various  other 
methods. 

*"Jour.  of  Infectious  Diseases,"  1913,  xn,  202. 


368  Hydrophobia,  Lyssa,  or  Rabies 

Williams  and  Lowden*  stained  Negri  bodies  by  one  of  the  follow- 
ing methods: 

(a)  Giemsa's  solution. — The  smears  are  fixed  in  methyl  alcohol  for  about  i 
minutes.     The  staining  solution  recommended  is  that  last  used  by  Giemsa: 

Azur  II.     Eosin 3.0 

Azur  II 0.8 

Glycerin  (Merck's  chemically  pure) 250.0 

Methyl  alcohol  (chemically  pure) 250.0 

Both  the  glycerin  and  methyl  alcohol  are  heated  to  6o°C.  The  dyes  ar< 
put  into  the  alcohol  and  the  glycerin  is  added  slowly,  stirring.  The  mixture  i: 
allowed  to  stand  at  even  temperature  over  night,  and  after  filtration  is  read> 
for  use.  At  the  time  of  use  one  drop  of  the  stain  is  added  for  every  cubic  centi 
meter  of  distilled  water  made  alkaline  by  the  addition  of  one  drop  of  a  i  pe 
cent,  solution  of  potassium  carbonate  to  10  cc.  of  the  water. 

The  stain  is  poured  on  the  slide  and  allowed  to  stand  for  from  one-half  to  threi 
hours.  The  longer  time  brings  out  the  structure  better  and  in  twenty-four  hour: 
well-made  smears  are  not  overstained.  After  the  stain  is  poured  off,  the  smea 
is  washed  in  running  tap  water  for  from  one  to  three  minutes  and  dried  witl 
filter-paper. 

By  this  method  the  "bodies"  are  stained  blue  and  the  central  bodies  an( 
chromatoid  granules  blue,  red  or  azure.  The  cytoplasm  of  the  nerve  cells  stain 
blue  also,  but  the  bodies  can  be  seen  distinctly  within  it.  For  diagnostic  purpose 
the  method  may  be  shortened  thus: 

Methyl  alcohol 5  minutes. 

Equal  parts  of  Giemsa  solution  and  distilled  water 10  minutes. 

(b)  The  eosin-methylene  blue  of  Mallory  (q.v.). 

The  smears  are  fixed  in  Zenkers'  solution  for  one-half  hour;  after  being  rinse( 
in  tap  water  they  are  placed  successively  in  95  per  cent,  alcohol  and  iodim 
for  one-quarter  hour,  95  per  cent,  alcohol  for  one-half  hour,  absolute  alcoho 
one-half  hour,  eosin  solution  20  minutes,  rinsed  in  tap  water,  methylene  blu< 
solution  15  minutes;  differentiated  in  95  per  cent,  alcohol,  lasting  one  to  fiv 
minutes  and  dried  with  filter-paper. 

With  this  method  the  cytoplasm  of  the  "bodies"  is  magenta,  light  in  the  smal 
bodies,  darker  in  the  larger;  the  center  bodies  and  chromatoid  granules  are  j 
very  dark  blue,  the  nerve-cell  cytoplasm  a  light  blue,  the  nucleus  a  darker  blui 
and  the  red  blood-cells  a  brilliant  eosin  pink. 

Harris f  uses  the  following  method  of  staining  Negri  bodies  tha 
seems  to  have  the  advantages  of  coloring  them  so  as  to  bring  out  thei: 
structure,  and  to  do  away  with  the  granular  precipitate  that  occur; 
in  most  other  methods. 

Smears  of  the  appropriate  material  are  made  upon  slides  and  fixed  by  thi 
application  of  methyl  alcohol  for  one  minute,  are  then  washed  with  water  t< 
remove  the  alcohol,  placed  for  from  one  to  three  minutes  in  an  old  saturatec 
solution  of  eosin  in  96  per  cent,  alcohol,  after  which  they  are  washed  for  two  o 
three  seconds  with  water  to  remove  the  excess  of  eosin.  This  stains  the  Negr 
bodies.  Counterstaining  is  effected  by  immersing  for  five  to  fif teen  _  second 
in  a  fresh  solution  of  Unna's  alkaline  methylene  blue,  after  which  there  is  a  brie 
washing  in  water,  decolorization  in  95  per  cent,  alcohol  and  then  the  usual  treat 
ment  with  absolute  alcohol,  xylol  and  balsam  if  the  preparation  is  to  be  covere( 
and  preserved,  or  the  spread  is  blotted  and  dried  if  to  be  examined  without  i 
cover.  The  whole  process  requires. less  than  five  minutes. 

Smears  that  have  been  dried  for  several  days  or  weeks  cannot  be  thus  stainec 
with  satisfaction.  The  older  the  eosin  solution  the  more  rapidly  and  intensely 
it  stains.  To  secure  the  best  results  it  should  not  be  less  than  two  months  old 
The  methylene  blue  should  not  be  more  than  a  week  or  two  old,  else  it  will  yielc 
an  objectionable  precipitate. 

*"  Jour,  of  Infectious  Diseases,"  1906,  in,  452. 
f  "Jour,  of  Infectious  Diseases,"  1908,  v,  566. 


Pathology  369 

Reichel  and  Engle*  stain  Negri  bodies  with  the  following: 

Sat.  ale.  sol.  methylene  violet.. . 10  cc. 

Sat.  ale.  sol.  fuchsin 7  drops. 

Sterile  water 40  cc. 

The  smears  of  cerebellum  or  hippocampus  are  fixed  with  absolute  alcohol  and 
ether  and  the  stain  poured  on,  heated,  poured  back  into  the  bottle,  again  poured 
on,  heated  and  poured  back  into  the  bottle,  this  being  done  three  times,  each 
time  for  about  half  a  minute.  Then  wash  in  water,  blot  and  examine.  To 
examine,  a  nerve-cell  is  found  with  the  low  power  and  then  examined  with  the 
high  power.  The  Negri  bodies  are  brick  red.  The  stain  soon  fades.  Smears 
kept  for  any  length  of  time  lose  the  staining  reaction. 

Luzzanif  gives  the  following  method  of  staining  Negri  bodies. 

The  tissue  to  be  stained  should  be  fixed  in  Zenker's  solution,  imbedded  in 
paraffine  and  cut  into  very  thin  slices.  Mann's  stain  is  used: 

i :  100  aqueous  solution  of  eosin 45  cc. 

i:  100  aqueous  solution  of  methylene  blue 35  cc. 

Distilled  water 100  cc. 

(The  solution  of  eosin  and  of  methylene  blue  should  be  kept  separately, 
and  only  mixed  and  diluted  at  the  time  of  using.  The  diluted  mixture  does 
not  keep  longer  than  some  days,  or  at  best,  a  few  weeks.) 

After  the  sections  are  cut,  they  are  fixed  to  the  slides  with  Mayer's  glycerin 
albumen,  the  paraffine  removed  with  xylol,  the  xylol  with  alcohol,  and  the 
alcohol  with  water.  The  stain  is  then  applied  for  some  minutes  after  which  the 
section  is  rapidly  washed  in  tap  water,  then  in  absolute  alcohol;  when  dehydrated 
in  the  absolute  alcohof,  they  are  washed  in  a  solution  of 

Absolute  alcohol 30  cc. 

Saturated  solution  of  caustic  soda  in  absolute  alcohol  5  drops. 

until  they  lose  the  blue  color  and  become  entirely  red.  They  are  then  given  a 
washing  in  absolute  alcohol,  plunged  into  tap  water  and  then  washed  with 
distilled  water  slightly  acidified  with  acetic  acid  until  they  turn  blue  again.  The 
final  steps  are  absolute  alcohol,  xylol  and  Canada  balsam.  The  Negri  bodies  are 
red,  the  cells  blue. 

The  method  should  be  as  applicable  for  smears  or  contact  spreads 
as  for  sections,  and  for  purposes  of  diagnosis  the  hippocampal  con- 
volution can  be  cut  across,  a  clean  side  touched  to  the  cut  surface  and 
removed.  Nerve-cells  adhere  to  the  glass  which  is  dried  and  treated 
as  though  it  had  an  adhering  section  of  tissue.  The  Negri  bodies  are 
best  seen  in  the  processes  of  the  nerve-cells. 

Pathology. — It  is  generally  supposed  that  the  activity  of  the  rabic 
virus  is  largely  confined  to  the  nervous  system,  and  that  from  the 
point  of  admission  to  the  body  it  ascends  the  peripheral  nerves  to 
effect  its  final  and  fatal  influence  upon  the  central  nervous  system. 
The  seat  of  inoculation  has,  therefore,  much  to  do  with  the  facility 
and  rapidity  with  which  the  symptoms  and  termination  come  on. 

When  the  virus  enters  through  the  skin  of  the  forearm  or  lower 
limb,  it  has  a  long  way  to  travel,  and  the  period  of  incubation  is  long; 
when  it  enters  about  the  face,  a  correspondingly  short  distance  to  go, 
and  a  correspondingly  brief  period  of  incubation.  The  occurrence 

*  Personal  communication. 

t"Ann,  de,  1'Inst,  Pasteur,"  1913,  xxxvn,  1039. 


37°  Hydrophobia,  Lyssa,  or  Rabies 

of  symptoms  is  accepted  as  evidence  that  the  central  nervous  system 
has  been  reached. 

When  as  in  experimental  inoculation  the  virus  is  at  once  placed  in 
the  central  nervous  system,  symptoms  do  not  at  once  develop,  hence 
it  is  concluded  that  not  only  must  the  essential  parasites  reach  the 
central  nervous  system,  but  they  must  do  so  in  sufficient  numbers 
before  enough  damage  can  be  done  to  produce  the  symptoms.  Under 
the  most  favorable  conditions  of  infection,  this  requires  about  six 
days. 

The  virus  is,  however,  not  confined  to  the  nervous  system  for  the 
saliva  is  infective,  and  the  salivary  glands,  pancreas,  and  perhaps 
other  glands  harbor  the  infective  agent.  How  it  reaches  these 
structures  has  not  yet  been  determined.  In  them  Negri  bodies  are 
present  but  whether  they  reach  the  glands  through  the  blood  or  by 
way  of  their  nervous  connections  is  not  known. 

There  is  no  morbid  anatomy  of  rabies.  Carefully  made  autopsies 
upon  the  bodies  of  rabid  human  beings  and  animals  show  nothing 
by  which  the  nature  of  the  disease  can  be  determined.  Most  inter- 
est naturally  centers  about  the  brain  and  spinal  cord  as  being  the 
chief  sources  of  disturbance  and  chief  seats  of  the  virus.  There  are, 
however,  so  few  changes  as  scarcely  to  merit  description.  In  some 
cases  the  meninges  are  distinctly  congested,  but  in  uncomplicated 
cases  there  is  no  meningitis  and  therefore  no  inflammatory  exudation. 

In  a  few  cases  there  may  be  scattered  minute  hemorrhages.  In 
many  cases  there  are  no  lesions. 

The  pathologic  histology  of  rabies  reveals  certain  fairly  constant 
lesions  described  in  the  next  section,  but  they  are  not  now  regarded 
as  characteristic  of  the  disease. 

Diagnosis  of  Rabies. — There  are  three  means  of  arriving  at  a 
diagnosis  of  rabies  in  cases  of  suspected  "mad-dogs." 

The  animal  having  been  killed,  its  head  is  cut  off  by  an  incision 
through  the  neck  at  some  distance  from  the  skull,  and  immediately 
taken  to  an  appropriate  laboratory  or  carefully  packed  in  plenty  of 
ice  and  sent  to  the  laboratory  by  express.  The  fresher  the  tissue 
received  by  the  laboratory  worker,  the  more  certain  his  results 
can  be. 

Carefully  opening  the  skull  of  the  dog,  the  brain  is  removed  to  a 
sterile  dish.  Good  sized  bits  of  tissue  are  taken  from  the  appropriate 
portions  of  the  brain  and  placed  in  glycerin  for  future  inoculation 
operations  if  necessary,  small  bits  of  the  same  tissue  are  spread  upon 
slides  according  to  the  "smear"  method  of  Williams  and Lowden,  or 
slides  may  be  spread  by  the  "adhesion"  method  of  Frothingham* 
who  makes  an  incision  into  the  brain,  lays  it  open  in  the  appropriate 
areas,  and  then  applies  the  flat  surface  of  a  perfectly  clean  slide  to 
the  flat  cut  surface  of  the  brain.  When  the  slide  is  lifted  up  (not 
slid  off),  nerve-cells  adhere  to  it,  in  which  the  Negri  bodies  may 
*  Jour.  Med.  Research,  1916,  xiv,  477. 


Inoculation  of  Rabbits  371 

later  be  found.  Other  parts  cut  from  the  appropriate  areas  of  the 
brain  tissue  are  placed  in  fixative  to  prepare  for  sectioning  should 
that  later  become  desirable. 

Williams  and  Lowden*  devised  a  new  technic  of  examination  for 
Negri  bodies  that  has  been  of  considerable  advantage  to  those  en- 
gaged in  looking  for  them  for  assisting  in  the  diagnosis  of  rabies,  as 
well  as  in  studying  the  bodies  themselves.  It  may  be  called  the 
" smear  method"  to  differentiate  it  from  the  older  and  less  certain 
"section  method."  Briefly,  the  method  is  as  follows: 

Glass  slides  and  cover-glasses  are  washed  thoroughly  with  soap  and 
water  and  heated  in  a  flame  to  get  rid  of  oily  substances.  A  small 
bit  of  the  gray  substance  of  the  brain  chosen  for  examination  is 
placed  upon 'one  end  of  a  slide,  a  cover-glass  placed  upon  it  and 
pressed  down  so  as  to  spread  out  the  nervous  tissue  in  a  thin  layer, 
when  the  cover  is  slowly  moved  to  the  opposite  end  of  the  slide 
spreading  out  the  nerve-cells  and  distributing  them  over  the  surface. 
The  tissues  selected  for  examination  should  come  from  at  least  three 
different  parts  of  the  gray  matter  of  the  central  nervous  system,  first, 
from  the  cortex  of  the  brain  in  the  neighborhood  of  the  fissure  of 
Rolando,  or  in  the  region  corresponding  to  it;  second,  from  Ammon's 
horn;  third,  from  the  cerebellum. 

The  smears  are  dried  in  the  air  and  then  stained  as  stated  above. 

Formerly  an  examination  of  the  spinal  sympathetic  ganglia  was 
made,  and  the  diagnosis  made  from  what  was  found  in  them.  This 
constitutes  the  least  important  and  most  rarely  pursued  form  of  diag- 
nostic procedure  at  the  present  time.  However,  we  will  suppose 
some  sympathetic  ganglia  secured.  The  remainder  of  the  animal's 
head  can  then  be  destroyed.  With  the  material  thus  secured  we 
make  the  following  diagnostic  tests: 

1.  Examination  for  the  Negri  bodies. 

2.  Inoculation  of  rabbits. 

3.  Examination  for  histological  changes  in  the  ganglia. 

1.  The  Negri  Bodies. — As  now  generally  conceded,  the  discovery 
of  these  bodies  in  the  cells  of  the  central  nervous  system  may  be 
taken  as  positive  evidence  of  the  existence  of  rabies  in  its  transmis- 
sible stage. 

2.  The  Inoculation  of  Rabbits. — This  is  only  necessary  in  highly 
suspicious  cases  in  which  no  Negri  bodies  are  found,  or  in  which  the 
investigator  is  not  satisfied  that  such  bodies  are  specific  indications 
of  the  disease. 

The  glycerinated  or  fresh  nervous  tissue  can  be  employed.  A  bit  of 
the  tissue  is  made  into  a  creamy  suspension,  under  aseptic  precau- 
tions, by  adding  physiological  salt  solution,  crushing  and  grinding 
in  a  small  agate  mortar.  When  it  is  ready  a  rabbit  is  anesthetized, 
the  hair  is  pulled  out  over  one  side  of  the  skull  (or  if  it  be  preferred, 

*  "Jour,  of  Infectious  Diseases,"  1906,  in,  452. 


37 2  Hydrophobia,  Lyssa,  or  Rabies 

the  skin  can  be  shaved),  the  scalp  is  washed  with  an  antiseptic 
solution  and  an  incision  about  an  inch  long  is  made  and  the  skull 
exposed.  With  a  small  trephine  a  button  of  bone  is  cut  out  and  the 
dura  exposed.  The  suspension  of  nervous  tissue  is  drawn  up  in  a 
sterile  hypodermic  syringe,  and  one  or  two  drops  of  it  injected  be- 
neath the  dura  mater  or  deeply  into  the  brain  tissue.  If  the  opera- 
tion be  successful  the  wound  heals  and  no  meningitis  follows,  but  at 
the  end  of  about  six  days  the  rabbit  becomes  paralyzed,  "dumb 
rabies."  Several  rabbits  should  be  simultaneously  inoculated  as 
should  a  single  rabbit  develop  meningitis,  through  accident  or  bad 
technic,  no  information  is  gained,  and  no  diagnosis  is  possible. 
The  rabid  rabbits  die  in  a  day  or  two  after  the  onset  of  the  palsy,  and 
Negri  bodies  can  be  found  in  the  brain  tissue,  which  is  infectious  for 
other  rabbits  in  endless  series. 

3.  The  Histological  Changes  in  the  Nervous  System. — These  are 
now  rarely  looked  for,  as  experience  has  shown  them  to  be  the  least 
reliable  means  of  making  the  diagnosis.  The  chief  changes  are  the 
"  tubercles  of  Babes,"*  which  consist  of  perivascular  collections  of 
cells,  and  collections  of  newly  formed  cells  about  the  ganglionic 
nerve-cells  of  the  brain  and  cord. 

Van  Gehuchten  and  Nelis,f  and  Ravenel  and  McCarthy J  have 
studied  these  lesions.  Ravenel  and  McCarthy  think  that  Babes 
gave  undue  prominence  to  the  rabid  tubercle,  which  consists  of  an 
aggregation  of  embryonal  cells  about  the  central  canal  of  the  cord, 
about  the  ganglionic  nerve-cells,  and  about  the  capillary  blood- 
vessels. They  think,  however,  that  the  lesions  of  the  nerve-ganglion 
cells  are  pathognomonic  if  taken  in  connection  with  the  clinical 
manifestations  of  the  disease.  The  specific  changes  consist  of  de- 
generation, chromatolysis  and  even  total  disappearance  of  the  nuclei 
of  the  ganglion  cells,  dilatation  of  the  pericellular  space,  and  invasion 
not  only  of  this  space,  but  also  of  the  nerve-cells  by  embryonal  cells, 
and  at  the  same  time  the  appearance  of  small  corpuscles  which  are 
hyaline,  brownish  and  in  part  metachromatic.  Spiller§  refused  to 
regard  these  lesions  as  pathognomonic  of  rabies  and  it  is  now  gen- 
erally conceded  that  they  are  not  specific  of  rabies,  and,  therefore, 
not  to  be  looked  upon  as  of  more  than  confirmatory  evidence  of  the 
disease. 

Virulence. — The  virus  of  rabies  is  variable  in  virulence  to  a 
marked  degree.  "Street  virus,"  or  that  obtained  from  rabid  dogs, 
is  so  variable  that  before  scientific  study  with  it  is  possible,  it  must 
be  standardized.  This  is  done  by  passage  through  rabbits,  the  tech- 
nic of  the  inoculation  being  the  same  as  that  given  in  the  section 
on  "Diagnosis."  After  being  passed  successively  from  rabbit  to 

*  Ann.  de  1'Inst.  Pasteur,  1896,  vi,  209. 
f  "Univ  med.  Mag./'  Jan.,  1901. 

"Archiv.  de  Biologie, "  1900,  xvi. 

"Pathological  Society  of  Philadelphia,"  March,  1901. 


Prophylaxis  373 

rabbit  from  twenty  to  thirty  times,  a  maximum  virulence  is  attained 
and  the  virus  is  said  to  be  "  fixed."  Pasteur  found  that  the  virulence 
of  the  nervous  tissue  was  diminished  by  inspissation,  by  drying  under 
aseptic  precautions  in  a  sterile  jar  over  calcium  chloride.  There  is 
some  doubt  whether  this  results  in  actual  diminution  in  the  virulence 
of  the  organisms  as  Pasteur  thought,  or  whether  the  virulence  is 
diminished  by  dilution,  i.e.,  by  effecting  the  destruction  of  many 
of  the  organisms.  There  seems  to  be  no  means  of  determining  this 
at  present.  The  diminution  of  virulence  is  in  proportion  to  the  length 
of  time  the  nervous  tissue  is  dried. 

Prophylaxis. — To  prevent  rabies,  means  must  be  devised  for 
preventing  dog-bites.  In  an  island  community  like  England,  rabies 
may  be  successfully  eliminated  by  destroying  all  animals  suspected 
of  having  the  disease,  muzzling  the  dogs  for  a  time,  and  denying 
admission  to  new  dogs  until  they  have  spent  a  long  enough  period 
in  quarantine  to  exclude  the  possibility  of  their  being  infected  with 
the  disease. 

Upon  continents  it  seems  unlikely  that  rabies  can  ever  be  com- 
pletely eradicated  as  it  is  not  only  a  disease  of  dogs,  but  also  of 
wolves,  foxes,  skunks  and  other  wild  animals  by  which  dogs  may  be 
bitten. 

However,  it  is  the  dog  that  is  the  common  distributor  and  to 
which  attention  must  be  directed. 

All  rabid  animals  should  at  once  be  killed,  and  all  others  known 
to  have  been  bitten  by  them  also  killed  so  soon  as  the  diagnosis  of 
rabies  in  the  first  animal  is  confirmed.  If  the  bitten  animals  cannot 
for  any  reason  be  killed,  they  should  be  carefully  confined  until  the 
incubation  period  is  long  past.  All  stray  dogs  and  cats  should  be 
destroyed  because  not  being  under  any  observation,  their  condition 
is  not  known.  Dogs  in  general  should  be  muzzled  when  abroad. 

Immunity  to  rabies  may  be  brought  about  in  human  beings  by 
the  method  of  active  immunization  given  below,  but  as  rabies  is  a 
somewhat  rare  disease  of  human  beings,  it  does  not  seem  worth 
while  to  advise  immunization  except  when  there  is  some  particular 
danger  of  its  occurrence.  Such  danger  obtains  when  human  beings 
have  been  attacked  and  bitten  by  rabid  animals  or  by  dogs  running 
at  large,  whose  health  is  a  matter  of  doubt.  Recovery  from  rabies 
in  human  beings  is  practically  unknown.  Any  individual,  therefore, 
that  is  bitten  under  suspicious  circumstances  may  be  in  danger  of 
developing  an  almost  certainly  fatal  malady.  This  is  not  to  be  con- 
strued to  mean  that  every  person  bitten  by  a  certainly  rabid  dog 
must  necessarily  contract  rabies,  for  there  are  accidents  and  cir- 
cumstances attending  the  transmission  of  diseases  of  infectious  na- 
ture, but  whether  certain  or  not,  the  danger  of  rabies  is  great  in 
such  cases  and  they  ought  to  receive  immediate  care  and  attention. 
Many  content  themselves  with  an  attempted  destruction  of  the 
introduced  virus  by  applying  the  actual  cautery,  or  caustics,  or 


374  Hydrophobia,  Lyssa,  or  Rabies 

powerful  germicides  to  the  wounds  made  by  the  dog's  teeth,  and 
Lambert  who  worked  upon  this* matter  experimentally  came  to  the 
conclusion  that  though  a  few  cases  might  thus  be  saved,  the  method 
was  too  unreliable  to  be  recommended.  The  long  period  of  incuba- 
tion of  human  rabies  (from  15  to  250  days  and  averaging  40  days) 
is  the  source  of  salvation  for  many  infected  persons,  for  it  makes  it 
possible  to  effect  immunization  during  that  period  and  so  inhibit 
the  development  of  the  disease  itself. 

Immunization  against  Rabies. — Pasteur*  observed  that  the  viru- 
lence of  the  virus  was  less  in  animals  that  had  been  dead  for  some  time 
than  in  those  just  killed,  and  by  experiment  found  that  when  the 
nervous  system  of  an  infected  rabbit  was  dried  in  a  sterile  atmos- 
phere its  virulence  attenuated  in  proportion  to  the  length  of  time 
it  was  kept.  A  method  of  attenuating  the  virulence  was  thus  sug- 
gested to  Pasteur,  and  the  idea  of  using  attenuated  virus  as  a  pro- 
tective vaccine  soon  followed.  After  careful  experimentation  he 
found  that  by  inoculating  a  dog  with  much  attenuated,  then  with 
less  attenuated,  then  with  moderately  strong  virus,  it  developed 
an  immunity  that  enabled  it  to  resist  infection  with  an  amount  of 
virulent  material  that  would  certainly  kill  an  unprotected  dog. 

It  is  remarkable  that  this  method,  based  upon  limited  accurate 
biologic  knowledge,  and  upon  experience  with  very  few  micro-organ- 
isms, should  find  absolute  confirmation  as  our  knowledge  of  im- 
munity, toxins,  and  antitoxins  progressed.  Pasteur  introduced  the 
unknown  poison-producers,  attenuated  by  drying  and  capable  of 
generating  only  a  little  poison,  accustomed  the  animal  first  to 
them  and  then  to  stronger  and  stronger  ones  until  immunity  was 
established. 

For  the  treatment  of  infected  cases  exactly  the  same  method  is 
followed  as  for  the  production  of  immunity.  Indeed,  the  treatment 
of  a  patient  bitten  by  a  rabid  animal  is  simply  the  production  of 
immunity  during  the  prolonged  incubation  period  of  the  affection, 
so  that  the  disease  may  not  develop.  The  patient,  to  be  successfully 
treated,  must  come  under  observation  early. 

The  Attenuation  Method. — To  protect  human  beings  from  the 
development  of  hydrophobia  after  they  have  been  bitten  by  rabid 
animals,  it  is  necessary  to  use  material  of  standard  or  known  viru- 
lence. This  can  be  prepared,  according  to  the  directions  of  Hogyes,f 
by  the  passage  of  virus  from  a  rabid  animal  through  from  21  to  30 
rabbits. 

For  this  purpose  some  of  the  hippocampal  tissue  of  the  dog  is  made  into  an 
emulsion  with  sterile  salt  solution  and  injected  subcutaneously  into  a  rabbit. 
As  soon  as  this  animal  dies,  itsspinalcord  is  removed,  a  similar  emulsion  made 
with  a  fragment  of  it,  and  a  second  rabbit  inoculated,  and  so  on  through  the 
series  until  a  standard  virulence  is  attained  and  the  virus  is  said  to  be  "fixed.'? 

*  "  Compt-rendudel' Acad.  de  Sciences  de  Paris,"  xcn,  i25Q;xcv,  1187,  xcvm, 
457,  1229;  ci,  765;  en,  459,  835;  cm,  777. 

t  See  Kraus  and  Levaditi,  "Handbuch  der  Immunitatsforschung,"  i. 


The  Attenuation  Method 


375 


It  has  a  much  higher  degree  of  virulence  than  the  "street  virus"  taken  from  the 
rabid  dog,  but  its  virulence  does  not  vary.  In  most  laboratories  the  "fixed 
virus"  is  obtained  from  other  laboratories  and  kept  passing  through  rabbits. 
In  this  manner  uniformity  of  dosage  and  virulence  is  most  easily  maintained 
The  technic  of  obtaining  the  rabbit's  cord  given  by  Oshida*  is  the  one  now 
generally  employed.  As  given  by  Stimson,f  it  is  performed  at  the  Hygienic 
Laboratory  as  follows:  "The  rabbit,  when  completely  paralyzed,  is  killed  with 
chloroform  and  nailed  to  a  board,  back  uppermost,  and  thoroughly  wetted 
down  with  an  aseptic  solution  (i  per  cent,  trikresol).  An  incision  is  made  through 
the  skin  from  the  forehead  nearly  to  the  tail  and  the  skin  laid  back  on  each 
side,  the  ears  being  cut  close  to  the  head.  An  area  i  inch  wide  is  seared  with 
a  hot  iron  around  the  occiput  and  nuchal  region  and  ear  openings.  The  skull 
is  then  transversely  divided  in  the  center  of  the  seared  areas  by  means  of  bone- 


Fig.  138. — Removal  of  the  spinal  cord  from  a  rabbit  (Stimson,  Bull.  No.  65, 
Hygienic  Laboratory). 

cutting  forceps.  The  neck  is  dissected  loose  from  the  skin  and  a  large  square  of 
sterile  gauze  is  inserted  beneath  it.  The  lumbar  region  is  dissected  up  for  a  few 
inches  and  a  similar  piece  of  gauze  placed  beneath  it.  Then  a  piece  of  telegraph 
wire  about  14  inches  long,  bent  into  a  handle  at  one  end  and  having  a  small 
wisp  of  cotton  twisted  about  the  other  end,  is  used  to  push  the  cord  out  of  its 
canal.  The  spine  is  steadied  by  a  pair  of  lion-jawed  forceps. 

An  assistant  catches  the  cord  with  forceps  as  it  emerges  from  the  cervical 
opening  and  lifts  it  out.  The  spinal  nerves  are  torn  off  during  this  procedure, 
and  the  membranes  stripped  off,  leaving  a  clean  sterile  cord.  A  silk  ligature  with 
one  long  end  is  placed  around  the  upper  end,  and  another,  just  below  the  middle 
of  the  cord,  which  is  then  cut  into  two  pieces  just  above  the  lower  ligature.  A 
small  piece  is  cut  off  of  the  upper  end  of  the  upper  portion  and  placed  in  a  tube 
of  bouillon,  which  is  incubated  as  a  test  for  sterility.  The  cords  are  hung  in  the 
drying  bottle  over  sticks  of  caustic  potash  or  calcium  chloride. 

The  longer  the  cord  dries,  the  more  the  virulence  of  the  micro- 
organisms attenuates. 

When  the  cord  has  reached  the  necessary  attentuation,  i  cm.  of 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1901,  xxrx,  Orig.,  988. 
t"  Facts  and  Problems  of  Rabies,"  Hygienic  Laboratory,  Bulletin  No.  65, 
June,  1910,  Washington,  D.  C. 


376  Hydrophobia,  Lyssa,  or  Rabies 

it  is  emulsified  with  3  cc.  of  sterile  0.8  per  cent,  salt  solution  and  is 
ready  for  use.  There  can  be  no  absolute  accuracy  of  dosage.  The 
injection  material  made  in  the  laboratory  under  strict  aseptic  pre- 
cautions can  be  used  with  perfect  safety  for  many  hours  subsequently 
if  kept  cold,  and  can  be  packed  in  ice  and  sent  by  express  to  the  phy- 
sician to  use  at  the  home  of  his  patients. 


Fig.  139. — Method  of  drying  the  spinal  cord  of  a  rabbit  for  the  purpose  of 
attenuation  (Stimson,  Bull.  No.   65,  Hygienic  Laboratory). 

As  the  transfer  of  the  cord  to  glycerin  preserves  the  virulence  for 
some  time  at  whatever  degree  it  had  when  so  transferred,  it  is  now 
customary  to  keep  on  hand,  in  glycerin,  in  the  laboratory,  spinal 
cords  of  rabbits  dried  one,  two,  three,  four  days,  and  so  on  through 
the  whole  series,  always  available  for  furnishing  vaccines  of  all  re- 
quired strengths,  independently  of  new  experimental  rabbits,  and 
also  makes  it  possible  for  one  rabbit  cord  to  furnish  material  for 
several  cases.  The  treatment  of  a  patient  bitten  by  a  rabid 
animal,  and  in  danger  of  acquiring  rabies,  requires  numerous  injec- 
tions with  material  of  varying  virulence,  as  shown  in  the  following 
tabulations: 


Scheme  for  Mild  Treatment 

PASTEUR'S  ORIGINAL  SCHEME  (Marx) 


377 


Light  schema 

ntense  schema 

Day  of  treatment 

Age  of 
dried 
cord 

Amount  of 
injected 
emulsion 

Day  of  treatment 

Age  of 
dried 
cord 

Amount  of 
injected 
emulsion 

First  

Days 

(14 
13 

12 
II 
IO 

9 

8 

'    6 
6 
5 
5 
4 
3 
5 
5 
4 
4 
3 
3 
5 
4 
3 

CC. 
3 
3 
3 
3 
3 
3 
3 
3 

2 
2 
2 
2 
2 
I 

2 
2 

2 
2 
2 
2 
2 

TTirof 

Days 
14 
.   13 

12 

ii 

10 

9 

8 

1 

I    6 
5 
5 
4 
3 
4 
3 
5 
5 
4 
4 
3 
3 
5 
4 
3 
5 
4 
3 

CC. 

3 
3 
3 
3 
3 
3 
3 
3 

2 
2 
2 
2 
2 
I 
2 
I 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 

Second  
Third 

Second       

Fourth  

Third  

Fifth  
Sixth 

Fourth 

Seventh 

Fifth 

Eighth 

Sixth 

Ninth  
Tenth  
Eleventh  
Twelfth  
Thirteenth 

Seventh  

Eighth  
Ninth  

Tenth 

Eleventh  

Fourteenth 

Twelfth  
Thirteenth 

Fifteenth  
Sixteenth 

Fourteenth 

Seventeenth.  .  .  . 
Eighteenth  

Fifteenth 

Sixteenth  
Seventeenth  
Eighteenth  
Nineteenth  
Twentieth  

Twenty-first  

(From  Bulletin  No.  65,  Hygienic  Laboratory,  June,  1910,  U.  S.  Public  Health 
and  Marine-Hospital  Service.) 

The  system  of  treatment  at  present  used  at  the  Hygienic  Labora- 
tory is  shown  in  the  following  tables: 

SCHEME  FOR  MILD  TREATMENT 


Amount  injected 

Amount   injected 

Day 

Cord 

Five 

One 

Day 

Cord 

Five 

One 

Adult 

to  ten 

to  five 

Adult 

to  ten 

to  five 

•, 

years 

years 

years 

years 

Injections 

CC. 

CC. 

CC. 

Injections 

CC. 

CC. 

CC. 

I  

8-7-6=3 

2-5 

2-5 

2  .O 

12.  ... 

4  = 

2-5 

2-5 

2-5 

2  

—  A  =  2 

2  •  5 

2  •  5 

j     r 

I  *? 

4  = 

2     C 

2  .  5 

2  •  5 

3  

T-        • 

4-3  =  2 

2.5 

2-5 

•  •  o 
2.0 

14  

3  = 

*  0 

2-5 

2.5 

2  .O 

4  

5  =  1 

2.5 

2.5 

2-5 

15.  ..  . 

3  = 

2-5 

2-5 

2  .O 

5  

4  =  1 

2.5 

2.5 

2-5 

16.... 

2  = 

2.5 

2.O 

I  .5 

6  

3  =  1 

2.5 

2.5 

2.O 

17.... 

2  = 

2-5 

2.0 

2-5 

7  

3  =  1 

2-5 

2-5 

2.O 

18.... 

4  = 

2-5 

2-5 

2-5 

8  

2  =  1 

2.5 

1.0 

£9  

3  = 

2.5 

2-5 

2-5 

9  

2  =  1 

2.5 

2  .O 

i  .  5 

20.  ... 

2  = 

2.5 

2-5 

2.0 

10  

5  =  1 

2.5 

2-5 

2-5 

21.  ... 

2  = 

2-5 

2-5 

2.0 

ii. 

5  =  i 

2-5 

2-5 

2-5 

378 


Hydrophobia,  Lyssa,  or  Rabies 

SCHEME  FOR  INTENSIVE  TREATMENT 


Amount  injected 

Amount  injected 

Day 

Cord 

Five 

One 

Day 

Cord 

Five 

One 

Adult 

to  ten 

to  five 

Adult 

to  ten 

to  five 

years 

years 

years 

years 

Injections 

cc. 

cc. 

cc. 

Injections 

cc. 

cc. 

cc. 

i  8-7-6=3 

2-5 

2-5 

2-5 

12..  .  . 

3=1 

2-5 

•2-5 

2  .0 

2  A  —  3  =  2 

2.5 

2.5 

2  .0 

13..  .  . 

3  = 

2  •  5 

2  •  5 

2  .O 

3  

5-4=2 

2-5 

2-5 

2-5 

14.  .  . 

2.5 

1.5 

2  .O 

4  

3  =  1 

2.5 

2-5 

2.0 

15.... 

2  = 

2.5 

2-5 

2.0 

5  

3  =  i 

2.5 

2.5 

2  .0 

16.... 

4  = 

2.5 

2.5 

2-5 

6  

2  =  1 

2.5 

2  .0 

i-5 

17.  .. 

3  = 

2-5 

2-5 

2-5 

7  

2  =  1 

2.5 

2-5 

2  .0 

18.... 

2  = 

2.5 

2-5 

2  .0 

8  

1  =  1 

2.5 

i-5 

I  .0 

19.... 

3  = 

2.5 

2-5 

2.0 

9  

5  =  I 

2-5 

2-5 

2-5 

20..  .  . 

2  = 

2-5 

2-5 

2-5 

10 

4=    I 
-1 

2     C 

2     C 

2     C 

21.. 

I  = 

2     C 

2     ^ 

2  .  O 

ii. 

4  =  1 

•  0 

2.5 

•         0 
2-5 

'  0 

2.5 

•  0 

•  J 

(From  Bulletin  No.  65,  Hygienic  Laboratory,  June,  1910,  U.  S.  Public  Health 
and  Marine-Hospital  Service.) 

The  Dilution  Method — Hogyes,*  of  Budapest,  believes  that  Pas- 
teur was  mistaken  in  supposing  that  the  drying  was  of  importance 
in  attenuating  the  virus,  and  thinks  that  dilution  is  the  chief  factor. 
He  makes  an  emulsion  of  rabbit's  medulla  (i  gram  of  medulla  to  10 
cc.  of  sterile  broth)  as  a  stock  solution,  to  be  prepared  freshly  every 
day,  and  uses  it  for  treatment,  the  first  dilution  used  being  i  :  10,000; 
then  on  succeeding  days  i  :  8000,  i  :  6000,  i  :  5000,  i  :  2000, 
i  :  1000,  i  :  500,  i  :  250,  i  :  200,  i  :  100,  and  finally  the  full  strength, 
i  :  10. 

Cabotf  found  the  dilution  method  attended  with  danger  to  the 
animal  immunized,  which  was  not  true  of  the  dried-cord  method  of 
Pasteur. 

The  Inspissation  Method. — A  new  method  of  carrying  out  the 
dilution  method,  suggested  by  Harris  and  Shackell,f  seems  to  be 
devoid  of  danger  to  the  patient  and  bids  fair  to  recommend  itself 
on  the  ground  of  greater  accuracy  than  former  methods.  It  depends 
upon  Shackell's  method  of  desiccation :{  ; 

The  material  to  be  dried  is  placed  in  the  bottom  of  a  Schubler's  vacuum 
desiccating  jar,  in  the  upper  part  of  which  is  a  separate  dish  containing  sulphuric 
acid.  The  temperature  is  reduced  by  placing  the  jar,  half  submerged,  in  a  salt 
and  ice  mixture,  and  after  thorough  solidification  of  the  material  has  resulted, 
a  rapid  vacuum  is  produced  by  a  Geryk  pump  to  less  than  2  mm.  of  mercury. 
During  the  process  of  desiccation,  the  temperature  in  the  lower  half  should  be 
kept  several  degrees  below  o°C.  Unless  the  sulphuric  acid  be  repeatedly  shaken 
to  prevent  saturation  with  water,  the  time  required  for  complete  desiccation 
will  be  unduly  prolonged. 

By  this  method  brains  and  cords  may  be  desiccated  in  toto,  with- 

*  "Acad.  des  Sciences  de  Buda-Pest,"  Oct.  17,  1897;  "Centralbl.  f.  Bakt.  u. 
Parasitenk.,"  1887,  n,  579. 

t ''Journal  of  Experimental  Medicine,"  1899,  vol.  iv,  No.  2. 
JLab.  Sec.  Amer.  Pub.  Health  Asso.,  Sept.  6,  1910. 


The  Inspissation  Method  379 

out  destruction  of  virulence,  in  from  twenty-four  to  thirty-six  hours. 
The  material  thus  dried  is  like  chalk  and  easily  pulverized.  It 
is,  however,  highly  hygroscopic  and  if  permitted  to  absorb  water 
becomes  leathery  and  loses  virulence  rapidly. 

In  a  later  paper  Harris*  found  that  the  more  thoroughly  and 
rapidly  the  material  is  frozen,  the  greater  will  be  the  amount  of 
virulence  remaining  after  desiccation.  A  new  method  suggested 
is  as  follows: 

"The  brain  or  cord  is  ground  in  a  porcelain  mortar,  with  the  addition  of 
water  drop  by  drop  until  a  thick  smooth  paste  is  formed.  Carbon  dioxide  snow 
is  then  collected  from  a  tank  in  the  ordinary  manner  and  is  added  in  small 
amounts  to  the  paste  which  should  be  stirred  thoroughly  meanwhile  to  prevent 
the  material  freezing  in  a  solid  mass.  Freezing  occurs  rapidly  and  when  complete 
the  material  is  very  brittle  and  easily  reducible  to  a  fine  powder.  During  the 
pulverization  more  snow  is  added  from  time  to  time  to  prevent  thawing.  When 
the  material  is  thoroughly  pulverized,  it  is  transferred  to  a  small  beaker  with  an 
excess  of  snow  and  placed  in  the  bottom  of  a  Schubler's  vacuum  jar  which  has 
previously  been  half  immersed  in  a  mixture  of  salt  and  ice  and  become  thoroughly 
cold.  A  beaker  of  sulphuric  acid  is  then  placed  on  wire  gauze  in  the  upper  part 
of  the  jar  in  such  manner  that  there  is  free  access  of  air  between  the  frozen  material 
and  the  sulphuric  acid.  The  acid  is  placed  in  the  upper  part  because  if  placed 
below,  it  soon  freezes  at  the  low  temperature.  The  vacuum  should  measure 
less  than  2  mm.  of  mercury.  During  desiccation  the  temperatures  should  not 
be  allowed  to  rise  above  —  is°C.  The  jar  should  be  rotated  gently  several 
times  daily  to  mix  the  water  and  the  acid.  A  single  brain  will  become  thoroughly 
dry  in  from  thirty-six  to  forty-eight  hours. 

The  object  in  thoroughly  pulverizing  the  virus  is  two-fold.  It 
results  in  a  more  complete  mixture,  so  that  all  parts  contain  an  equal 
amount  of  virulence.  Secondly,  it  permits  of  more  rapid  drying 
and  an  easy  transfer  into  smaller  containers  for  subsequent  tests. 
To  avoid  any  absorption  of  moisture,  the  dry  powder  is  transferred 
from  the  beaker  to  small  glass  tubes  the  ends  of  which  are  sealed  in 
a  flame.  The  transfer  is  effected  in  a  moisture-free  atmosphere  by 
covering  the  top  of  the  beaker  with  rubber  dam  held  in  place  by  ad- 
hesive strips.  A  small  puncture  is  made  in  the  rubber  large  enough 
to  admit  the  tube,  and  through  this  the  tubes  are  inserted  and  filled. 
From  20  to  100  mg.  is  a  convenient  amount  put  into  each  tube.  If 
the  tube  has  a  diameter  of  4  mm.,  each  millimeter  of  powder  will 
weigh  approximately  2  mg. 

Harris  believes  that  the  use  of  desiccated  virus  in  anti-rabic  im- 
munization of  animals  and  persons  offers  many  advantages  over 
other  methods. 

Harrisf  reports  that  182  patients  have  been  injected  with  the 
virus  thus  prepared  for  the  purpose  of  immunizing  them  against 
hydrophobia.  No  deaths  have  occurred  and  no  complications  de- 
veloped. It  is  thus  to  all  appearances  a  safe  and  efficient  method 
and  is  especially  economical  to  the  laboratory  in  time,  labor  and 
money.  Material  can  be  prepared  two  or  three  times  a  year  and 
put  aside  in  the  cold  to  be  used  only  when  needed  and  as  one  rabbit 

*  "Jour,  of  Infectious  Diseases,"  1912,  x,  369. 
f  "Jour,  of  Infectious  Diseases,"  1913,  xm,  155. 


380  Hydrophobia,  Lyssa,  or  Rabies 

furnishes  enough  material  to  immunize  20-25  patients,  the  initial 
cost  is  negligible.  The  work  can  be  undertaken  in  any  hospital  or 
municipal  laboratory  without  increasing  the  staff  or  the  expense. 
To  be  able  to  prepare  at  one  time  enough  material  for  from  six  to 
twelve  months'  use  and  to  have  this  always  ready  for  any  number  of 
patients  is  such  a  lessening  of  labor  and  anxiety  as  only  those  who 
have  followed  the  classic  method  of  drying  cords  can  appreciate. 

If  the  conclusion  of  Harvey  and  McKendrick*  be  correct,  and 
"the  immunizing  power  of  any  given  portion  of  a  rabies  cord  is  a 
function  of  the  unkilled  remnant  of  the  rabies  virus  which  is  con- 
tained in  that  cord,"  one  should  be  able  to  find  out  with  mathe- 
matical certainty  how  many  minimum  infective  doses  will  produce 
a  definite  degree  of  immunity.  For  this  purpose  they  suggest  that 
the  virulence  of  the  virus  is  expressed  in  "units,"  one  unit  being 
the  smallest  amount  which,  when  injected  intra-cerebrally  into  a 
full-grown  rabbit,  will  produce  paresis  on  the  seventh  day. 

Specific  Treatment. — Babes  and  Leppf  thought  that  the  serum 
of  animals  that  had  received  repeated  injections  of  the  crushed 
nervous  tissue  of  rabid  animals  was  neutralizing  or  destructive  to 
the  rabies  virus  in  vitro,  called  it  "antirabic  serum,"  and  believed 
that  it  conferred  a  defensive  power  upon  other  animals.  Marie,  J 
however,  found  it  to  be  a  simple  neurotoxic  serum  and  inert  in  its 
action  upon  the  virus.  It  is  never  used  in  the  treatment  of  rabies, 
at  present. 

*"  Theory  and  Practice  of  Anti-rabic  Immunization,"  Calcutta,  1907. 

t  "Ann.  de  1'Inst.  Pasteur,"  1889,  in. 

J"Compt.-rendu  Soc.  Biol.,"  June  18,  1904,  LVI,  p.  1030. 


CHAPTER  VI 
ACUTE  ANTERIOR  POLIOMYELITIS 

ACUTE  anterior  poliomyelitis,  atrophic  spinal  paralysis,  infantile 
palsy,  "spinale  Kinderlahmung,"  is  an  acute  infectious  disease, 
largely  confined  to  the  first  three  years  of  life,  and  characterized  by 
fever,  destruction  of  cells  in  the  gray  matter  of  the  central  nervous 
system,  palsy  and  rapid  atrophy  of  the  palsied  muscles.  It  is  of 
sporadic  and  occasionally  of  epidemic  occurrence  in  all  parts  of  the 
world.  Although  infectious,  its  transmissibility  is  so  slight  as  to 
make  contagiousness  a  matter  of  doubt. 

The  essential  cause  is  in  doubt,  though  it  is  possible  that  it  is  a 
minute  coccoid  organism  that  may  be  capable  of  artificial  cultivation. 
It  is  certain  that  there  is  an  infectious  agent  and  that  it  is  filterable 
through  the  Berkefeld  filters.  Probably  the  best  account  of  the 
history  and  epidemiology  of  the  disease  has  been  compiled  by 
Wickman.* 

The  disease  was  investigated  bacteriologically  by  various  workers, 
and  it  went  through  the  usual  experience  of  having  various  micro- 
organisms isolated  and  described,  to  be  afterward  abandoned  as 
accidental  and  unimportant  agents.  The  modern  studies  of  the  sub- 
ject, by  modern  methods  of  investigation,  were  begun  by  Landsteiner 
and  Popper. f  Their  method  of  procedure  was  to  emulsify  the 
spinal  cord  of  a  fatal  case  of  the  disease,  in  a  nine-year-old  child,  in 
physiological  salt  solution,  and  inject  it  into  the  peritoneal  cavities 
of  monkeys.  One  monkey  became  ill  and  died  on  the  eighth  day; 
the  other  became  paralyzed  on  the  seventeenth  day  after  the  inocu- 
lation. A  similar  emulsion  of  the  cord  of  the  paralyzed  monkey 
failed  to  infect  other  monkeys  into  which  it  was  injected.  Knopfel- 
macher,J  and  Strauss  and  Huntoon§  were  also  able  to  infect  one 
monkey  with  human  virus,  but  could  carry  the  infection  no  further. 

Flexner  and  Lewis  ||  made  careful  experiments  upon  81  monkeys 
inoculated  with  the  disease.  They  found  the  incubation  period  to 
vary  from  4  to  33  days,  the  average  being  9.82  days.  During  this 
period  there  were  prodromal  symptoms  such  as  nervousness  and 
excitability,  fatigue,  tremor  of  the  face  and  limbs,  shifting  gaze 

"Beitrage  zur  Kentniss  der  Heine-Medinischen  Krankheit,"  Berlin,  1907. 
t "Zeitschrif t  fur  Immunitatsforschung,"  1909,  n,  377. 

"  Med.  Klin.,"  1909,  v,  1671. 
§"New  York  Med.  Jour.,"  1910,  xci,  64. 

II  "Journal  of  the  Amer.  Med.  Assoc.,"  1909,  LIII,  1639,  and  "Jour.  Medical 
Research,"  1910,  xn,  227. 


382  Acute  Anterior  Poliomyelitis 

when  the  attention  was  attracted,  and  a  wrinkled  and  mobile  rather 
than  smooth  and  placid  face.  The  onset  of  the  disease  is  sudden, 
with  or  without  the  given  signs,  and  consists  of  paralysis.  In  gen- 
eral, any  of  the  larger  voluntary  muscle  groups  may  be  affected; 
other  groups  may  be  weak  or  partially  paralyzed.  The  paralysis 
may  be  of  all  grades  of  completeness.  There  may  be  some  anes- 
thesia; occasionally  there  was  evidence  of  pain.  The  animals  may 
die  or  they  may  recover.  In  the  latter  case  the  paralysis  some- 
times entirely  disappears;  more  frequently  it  persists  and  the  para- 
lyzed member  gradually  stiffens  and  is  deformed  by  contractures. 

In  the  dead  monkeys,  or  those  that  were  killed  for  study,  the 
chief  lesions  were  in  the  gray  matter  of  the  central  nervous  sys- 
tem and  consisted  of  edema,  diffuse  livid  injections  of  the  blood- 
vessels and  punctiform  and  pin-head-sized  hemorrhages.  When 
healing  sets  in,  the  lesions  are  firmer,  paler,  non-circumscribed,  and 
raised  somewhat  above  the  level  of  the  surrounding  gray  and  white 
matter. 

The  chief  histological  changes  were  also  in  the  gray  matter  es- 
pecially in  the  cord,  where  they  occurred  in  either  the  anterior  or 
posterior  horns,  but  more  frequently  and  more  extensively  in  the 
anterior  horns.  There  was  a  high  degree  of  cellular  infiltration  of 
the  perivascular  spaces,  edema  of  the  spaces,  and  hemorrhage  into 
the  spaces.  From  the  spaces  the  cells  often  passed  into  the  ground 
substance.  But  independent  foci  of  small  cells,  edema  and  hemor- 
rhage also  existed  in  the  nervous  tissue.  The  nerve  cells  often 
showed  degeneration  which  consisted  of  hyaline  transformation 
and  necrosis  leading  to  loss  of  the  tigroid  substance,  cell-processes, 
nuclei,  etc.  Often  the  cell  was  surrounded  by  lymphocytes  or  in- 
vaded by  polymorphonuclear  leukocytes.  Sometimes  the  nerve- 
cells  had  disappeared  and  the  leukocytes  taken  their  places.  Ulti- 
mately, a  part  of  the  nervous  elements  would  be  removed  and  re- 
placed by  an  indefinite  cellular  tissue,  containing  many  compound 
granular  corpuscles. 

The  monkeys  were  infected  by  various  methods,  the  first  being 
the  direct  inoculation  of  the  brain  by  a  needle  introduced  through 
the  opening  made  by  a  small  trephine.  They  found,  however, 
that  the  virus  readily  finds  its  way  to  the  nervous  system  when 
introduced  subcutaneously,  and  less  readily  when  introduced 
intraperitoneally.  The  blood  of  the  infected  animal  contains  the 
virus  at  the  beginning  of  the  attack  but  how  richly  was  not  deter- 
mined. The  crebro-spinal  fluid  also  contains  it  at  the  time  the 
palsy  appears.  The  vaso-pharyngeal  mucosa  also  contains  it,  and 
can  convey  it  to  other  animals. 

The  virus  readily  passed  through  Berkefeld  filters,  and  the  clear 
filtrate  thus  obtained,  when  injected  into  monkeys  by  the  intra- 
cerebral  or  subcut'aneous  routes,  regularly  produced  the  disease 
in  an  infectious  form  so  that  it  was  clear  that  the  lesions  were  in- 


Bacteriology 


383 


f  ectious  and  not  toxic  in  character  though  brought  about  by  filtered 
fluid. 

The  virus  resists  freezing  but  is  readily  destroyed  by  heating  to 
45°-5o°C.  for  half  an  hour. 

Various  attempts  were  made  by  Kraus  and  Wernicke,*  Lentz 
and  Huntemiillert  and  Marks {  to  infect  rabbits  with  the  virus,  but 


Fig.  140. — Micro-organism  causing  epidemic  poliomyelitis.  3,  Separate  glo- 
boid  bodies,  X  1000;  4,  aggregated  masses  ofgloboid  bodies,  X  1000;  5,  chains 
and  pairs  of  globoid  bodies,  X  1000;  6,  chains  of  globoid  bodies  compared  with 
Streptococcus  pyo genes,  X  1000;  7,  agar  fragment  showing  pairs  of  globoid  bodies 
compared  with  Streptococcus  pyogenes,  X  1000  (Flexner  and  Noguchi,  in 
Journal  of  Experimental  Medicine). 

though  some  successes  were  reported,  there  seems  to  be  no  develop- 
ment in  the  rabbit  of  lesions  or  disturbances  resembling  the  char- 
acteristic lesions  and  symptoms  of  acute  anterior  poliomyelitis  in 
man  and  the  monkey. 

*  "Deutsche  med.  Wochenschrift,"   1909,  xxxv,  1825;  1910,  xxxvi,  693. 

t  "Zeitschrift  fur  Hygiene,"  1910,  LXVI,  481. 

J"Jour.  Exp.  Med.,"  1911,  xiv,  116. 


384  Acute  Anterior  Poliomyelitis 

In  1912,  Rosenau  and  Brues*  reported  that  in  50  per  cent,  of  their 
experiments,  the  virus  of  acute  anterior  poliomyelitis  was  trans- 
mitted from  monkey  to  monkey  by  the  bite  of  the  stable  fly  Stomoxys 
calcitrans,  and  expressed  the  belief  that  it  was  a  biological  and  not  a 
mechanical  transfer,  and  that  the  virus  underwent  some  change  and 
development  in  the  flies.  These  results  were  confirmed  by  Ander- 
son and  Forst,f  but  failed  to  be  confirmed  by  other  workers  and 
later  could  not  be  successfully  repeated  by  the  same  investigators. 

Howard  and  ClarkJ  worked  over  the  subject  of  transmission  of 
the  disease  by  insects,  and  investigated  the  house-fly  Musca  domes- 
tica;  the  bed-bug,  Cimex  lectularius;  the  lice,  Pediculus  capitis  and 
Pediculus  vestimenti;  various  mosquitoes,  Culex  pipiens,  Culex 
solicitans  and  Culex  cantator,  and  found  that  only  one  of  these 
insects,  the  common  house-fly,  Musca  domestica,  can  carry  the  virus 
in  an  active  state  for  several  days  both  upon  the  surface  of  its 
body  and  in  its  gastro-intestinal  tract.  None  of  the  suctorial 
insects  withdrew  the  virus  with  the  blood  of  the  infected  monkeys 
to  which  they  were  applied. 

Flexner  and  Noguchi§  made  experiments  upon  the  cultivation 
of  the  micro-organism  supposed  to  be  the  infective  agent.  The 
technic  employed  was  much  like  that  employed  for  the  cultivation 
of  Treponema  pallidum  (q.v.),  and  resulted  in  an  undoubted  quan- 
titative increase  in  the  infectiveness  of  the  virus.  Further,  they 
were  now  able,  for  the  first  time,  to  describe  an  organism  that 
might  be  the  specific  infectious  agent.  It  is  a  globoid  body  meas- 
uring from  0.15-0.3  ju,  arranged  in  pairs,  chains  and  indefinite  masses. 
Its  small  size  makes  it  barely  visible  and  able  to  penetrate  the  pores 
of  the  Berkefeld  filters. 

This  organism  they  were  able  to  stain  both  by  the  methods  of 
Giemsa  and  Gram.  Having  come  to  recognize  it  in  the  culture, 
they  were  subsequently  able  to  find  it  in  sections  of  tissue  from  the 
lesions  of  poliomyelitis,  and  conclude  that  "The  micro-organism 
exists  in  the  infectious  and  diseased  organs;  it  is  not,  so  far  as  is 
known,  a  common  saprophyte,  or  associated  with  any  other  patho- 
logical condition;  it  is  capable  of  reproducing,  on  inoculation,  the 
experimental  disease  in  monkeys,  from  which  animals  it  can  be  re- 
covered in  pure  culture.  And  besides  these  classical  requirements, 
the  micro-organism  withstands  preservation  and  glycerination  as 
does  the  ordinary  virus  of  poliomyelitis  within  the  nervous  organs. 
Finally,  the  anaerobic  nature  of  the  micro-organism  interposes  no 
obstacle  to  its  acceptance  as  the  causative  agent,  since  the  living 
tissues  are  devoid  of  free  oxygen  and  the  virus  of  poliomyelitis  has 
not  yet  been  detected  in  the  circulating  blood  or  cerebro-spinal  fluid 

*  "Monthly  Bull,  of  the  State  Board  of  Health  of  Massachusetts,"  1912,  vn, 
314- 

"Public  Health  Reports,"  1913,  xxvm,  833. 
:"Jour.  Exp.  Med.,"  1912,  xvi,  850. 
§  "Jour.  Exp.  Med.,"  1913,  xvm,  461. 


Bacteriology  385 

of  human  beings,  in  which  the  oxygen  is  less  firmly  bound;  nor  need 
it,  even  should  the  micro-organism  be  found  sometimes  to  survive 
in  these  fluids." 

From  these  discoveries  it  is  now  certainly  well  established  that 
acute  anterior  poliomyelitis  is  an  infectious  disease,  occasioned  by 
a  minute  anaerobic  organism,  of  globoid  form,  capable  of  resisting  the 
bactericidal  effects  of  glycerin  for  months,  and  capable  of  passing 
through  the  pores  of  a  Berkefeld  filter.  When  nervous  or  other 
tissue  containing  it,  or  pure  cultures  of  it,  are  introduced  into  the 
nervous  tissue  or  into  the  subcutaneous  tissues  of  certain  animals, 
of  which  the  monkey  is  the  chief  one,  the  disease  is  readily  induced. 

The  mode  of  transmission  remains  to  be  discussed.  From  the 
failure  of  those  who  continued  the  insect  experiments  to  achieve 
continued  success,  and  because  of  the  short  time  the  infectious  agents 
are  in  the  blood — only  the  first  few  days — and  the  small  number  that 
seem  to  be  there,  it  is  well  to  assume  that  insects  play  a  doubtful 
role,  unless  it  be  the  common  house-fly,  Musca  domestica. 

Flexner  and  Clark*  have  shown  that  when  the  virus  is  introduced 
into  the  upper  nasal  mucosa  in  monkeys  its  propagation  can  be 
followed  from  the  olfactory  lobes  of  the  brain  to  the  medulla  oblongata 
and  spinal  cord.  Since  the  virus  can  thus  find  its  way  from  the  nasal 
mucosa  to  the  deeper  nervous  tissues,  they  hold  the  opinion  that 
it  is  through  this  avenue  that  infection  commonly  takes  place. 

During  the  disease,  the  infectious  agents  are  upon  the  nasal 
mucosa,  they  may  be  discharged  from  the  surface  into  the  atmos- 
phere,- and  inhalation  by  others  may  be  the  means  of  infection.  It 
is  also  not  impossible  that  house-flies  first  visiting  the  nose  of  an 
infected  sleeping  child,  and  then  some  other  sleeping  child,  may  carry 
the  organisms. 

One  attack  of  the  disease  confers  immunity,  and  experimental 
immunization  can  be  effected  by  a  succession  of  doses  beginning 
with  great  dilutions  and  ascending  to  greater  concentrations  like 
the  Hogyes  method  in  rabies,  but  as  the  disease  comes  on  without 
a  preliminary  dog-bite,  and  as  the  period  of  incubation  is  short,  and 
as  our  first  knowledge  of  it  coincides  with  the  appearance  of  the 
paralysis  when  the  damage  is  already  done,  no  practical  utilization 
can  be  made  of  our  knowledge  of  the  facts  of  immunity  to  the 
disease  at  the  present  time. 

*  "Proc.  Soc.  Exper.  Biol.  and  Med.,"  191 2-13,  x2 1. 
25 


CHAPTER  VII 
CEREBRO-SPINAL  MENINGITIS 

DIPLOCOCCUS    INTRACELLULARIS    MENINGITIDIS    (WEICHSELBAUM) 

General  Characteristics. — A  minute  non-motile,  non-flagellate,  non-sporog- 
enous,  non-chromogenic,  non-liquefying,  aerobic,  pathogenic  coccus,  staining 
by  ordinary  methods,  but  not  by  Gram's  method. 

Acute  cerebro-spinal  meningitis  may  be  secondary  to  various 
more  or  less  well-localized  infections  when  it  depends  upon  such 
micro-organisms  as  may  be  carried  by  accident  to  the  meninges. 
Among  these  may  be  mentioned  pneumococci,  staphylococci,  strep- 
tococci, Bacillus  influenzas,  B.  typhosus,  B.  coli,  B.  mallei,  B.  pestis 
and  others. 

In  addition  to  these  cases,  however,  there  are  numerous  cases  of 
primary  infection  of  the  membranes,  either  sporadic  or  epidemic  in 
occurrence.  Such  constitute  the  disease  known  as  cerebro-spinal 
fever,  epidemic  cerebro-spinal  meningitis,  or  u  spotted  fever"  It  is  a 
very  dangerous  febrile  malady,  characterized  by  high  temperature, 
an  irregular  exanthem,  early  meningitis,  a  moderate  degree  of  con- 
tagion, and  a  high  mortality.  The  cause  of  this  infection  is  a 
specific  organism  known  as  the  meningococcus,  or  Diplococcus  intra- 
cellularis  meningitidis. 

As  early  as  1887  Weichselbaum*  carefully  described  a  diplococcus 
found  in  6  cases  of  cerebro-spinal  meningitis  that  may  have  been 
identical  with  one  found  by  Leichtensternf  in  1885  in  the  purulent 
exudate  of  a  case  of  meningitis,  and  with  a  coccus  observed  as 
early  as  1884  by  Celli  and  Marchiafava.J  Weichselbaum's  studies 
and  description  of  this  coccus  seem  to  have  attracted  but  little 
attention  at  first,  and  references  to  them  are  but  brief  in  most  of 
the  text-books.  The  prevailing  opinion  was  that  its  occurrence  in 
cerebro-spinal  meningitis  was  accidental,  as  inoculations  into  ani- 
mals showed  its  pathogenic  power  to  be  very  limited.  The  careful 
studies  of  Jager,§  Scherer,||  Councilman,  and  Mallory  and  Wright** 
(embracing  55  cases,  in  which  the  cocci  were  found  by  culture  or 
by  microscopic  examination  in  38),  and  of  Flatten,ft  Schneider,ff 
Rieger,f|  Schmidt,  ff  Goppert,ff  Flugge,ft  von  Lingelsheim,ff 

*  "  Fortschritte  der  Med.,"  x,  18  and  19. 
t  "Deutsche  med.  Wochenschrift,"  1885. 
j  "Gazette  degli  Ospedali,"  1884,  vin. 

§  "Zeitschrift  fur  Hygiene,"  xix,  2,  351. 

||  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1895,  xvn,  13  and  14. 

*  "Amer.  Jour.  Med.  Sci.,"  March,  1898,  vol.  cxv,  No.  5. 
ft  "Klinisches  Jahrbuch,"  1906. 

386 


Identification  387 

Besredka*  Flexnerf  and  others  have,  however,  shown  the  diplo- 
coccus  of  Weichselbaum  to  be,  without  doubt,  the  specific 
organism. 

Distribution. — The  distribution  of  Diplococcus  intracellularis 
in  nature  is  as  yet  unknown.  It  has  been  found  in  cerebro-spinal 
meningitis  by  those  who  have  looked  for  it,  twice  has  been  found 
in  the  nose  in  coryza  by  Scherer,  has  been  found  in  the  conjunctiva 
by  Carl  FrankelJ  and  Axenfeld,§  and  in  the  purulent  discharges 
of  rhinitis  and  otitis  by  Jager.|| 

Morphology. — The  micro-organism  is  a  biscuit-shaped  dip- 
lococcus  having  a  great  resemblance  to  the  gonococcus.  This  re- 
semblance is  further  increased  by  the  fact  that  the  cocci  are  usually 
found  inclosed  in  the  protoplasm  of  the  leukocytes.  Weichselbaum, 


'I 


Fig.  141. — Meningococcus  in  spinal  fluid  (from  Hiss  and  Zinsser,  "Text-Book 
of  Bacteriology,"  D.  Appleton  &  Co.,  Publishers). 

by  whom  this  was  first  observed,  found  it  constant  in  sections  of 
the  brain  and  its  membranes,  though  in  the  exudate  of  the  disease 
a  good  many  free  cocci  may  be  observed.  It  was  this  peculiar 
relationship  to  the  cells  that  led  Weichselbaum  to  name  the  organ- 
ism Diplococcus  intracellularis.  Many  of  the  cocci  inclosed  in  the 
cells  are  apparently  dead  and  degenerated,  as  they  stain  badly  and 
do  not  grow  when  the  pus  is  transferred  to  culture-media. 

Identification.— Carl  Frankel,  in  discussing  the  micro-organ- 
ism, points  out  that  its  morphologic  peculiarities  have  much  in 
common  with  the  pneumococcus,  so  that  the  most  refined  methods 
of  differentiation  should  always  precede  a  positive  determina- 
tion. Its  resemblance  to  the  gonococcus  should  also  be  kept  in 
mind. 

k  Perhaps   the   greatest   difficulty   obtains   in   making   a   certain 
*  "Annales  de  1'Inst.  Pasteur,"  1906,  xx,  4. 
t  "Jour.  Exp.  Med.,"  1906-07. 
:  "Zeitschrift  fiir  Hygiene,"  June  14,  1899. 
§  Lubarsch  and  Oestertag,  "Ergebnisse  der  allg.  Path.  u.  path.  Anat.,"  in, 

s-  573- 

"Deutsche  med.  Wochenschrift,"  1894,  S.  407. 


388  Cerebro-spinal  Meningitis 

differentiation  between  the  meningococcus  and  Micrococcus  catar- 
rhalis  (q.v.),  especially  when  such  investigations  are  directed  toward 
discovering  the  former  organism  in  the  nasal  discharges.  This  can- 
not be  done  by  microscopic  examination,  but  must  be  achieved 
through  cultivation  of  the  organisms  and  observation  of  the  cultures. 
Micrococcus  catarrhalis  grows  well  upon  nearly  all  culture-media; 
meningococci,  very  sparsely  except  upon  special  media.  The 
former  organism  grows  fairly  well  at  room  temperatures  (2o°C.  or 
less);  the  latter,  only  at  25°C.  and  above.  The  colonies  of  the 
former  are  coarsely  granular;  those  of  the  latter,  finely  granular. 

Staining. — The  organism  is  easily  stained  with  the  usual  aqueous 
solutions  of  the  anilin  dyes.  It  does  not  stain  by  Gram's  method. 

For  staining  the  meningococcus  the  method  of  Pick  and  Jacob- 
sohn*  is  highly  praised  by  Carl  Frankel,  who  modifies  it  by  adding 
three  times  as  much  carbol-fuchsin  as  is  recommended  in  the 
original  instructions,  which  are  as  follows:  Mix  20  cc.  of  water 
with  8  drops  of  saturated  methylene-blue  solution;  then  add  45  to 
50  drops  of  carbol-fuchsin.  Allow  the  fluid  to  act  upon  the  cover- 
glass  for  five  minutes.  The  cocci  alone  are  blue,  all  else  red. 

Isolation. — The  organism  can  be  secured  for  cultivation  either 
from  the  purulent  matter  of  the  exudate  found  at  autopsy,  or  from 
the  fluid  obtained  by  lumbar  puncture.  To  obtain  this  fluid 
Parkf  gives  the  following  directions:  "The  patient  should  lie  on 
the  right  side  with  the  knees  drawn  up  and  the  left  shoulder  de- 
pressed. The  skin  of  the  patient's  back,  the  hands  of  the  operator, 
and  the  large  antitoxin  syringe  should  be  sterile.  The  needle 
should  be  4  cm.  in  length,  with  a  diameter  of  i  mm.  for  children, 
and  larger  for  adults.  The  puncture  "is  generally  made  between 
the  third  and  fourth  lumbar  vertebrae.  The  thumb  of  the  left  hand 
is  pressed  between  the  spinous  processes,  and  the  point  of  the 
needle  is  entered  about  i  cm.  to  the  right  of  the  median  line  and 
on  a  level  with  the  thumb-nail,  and  directed  slightly  upward  and 
inward  toward  the  median  line.  At  a  depth  of  3  or  4  cm.  in  children 
and  7  or  8  cm.  in  adults  the  needle  enters  the  subarachnoid  space, 
and  the  fluids  flow  out  in  drops  or  in  a  stream.  If  the  needle  meets 
a  bony  obstruction,  withdraw  and  thrust  again  rather  than  make 
lateral  movements.  Any  blood  obscures  microscopic  examination. 
Adults,  not  too  ill,  may  sit  upon  a  chair  or  upon  the  edge  of  the  bed 
while  the  spinal  puncture  is  made,  as  shown  in  Kolmer's  illustration. 
The  fluid  is  allowed  to  drop  into  sterile  test-tubes  or  vials  with  sterile 
stoppers.  From  5  to  15  cc.  should  be  withdrawn.  No  ill  effects 
have  been  observed  from  the  operation." 

In  making  a  culture  from  this  fluid  Park  points  out  that,  as 
many  of  its  contained  cocci  are  dead,  a  considerable  quantity  of  the 
fluid  (say  about  i  cc.)  must  be  used. 

*  rcBerlinef  klin.  Wochenschrift,"  1896,  811.  __ 

t  "Bacteriology  in  Medicine  and  Surgery,"  Philadelphia,  1899,  p.  364. 


Cultivation 


389 


The  cocci  have  also  been  cultivated  from  the  nasal  discharges 
in  6  cases  studied  by  Weichselbaum,  and  in  18  studied  by 
Scherer.  Elser*  has  isolated  the  organism  from  the  circulating 
blood  of  patients  suffering  from  epidemic  cerebro-spinal  fever.  To 
determine  the  presence  of  the  coccus  in  the  nasal  discharges  where 
other  similar  cocci  may  be  present,  Gram's  stain  may  be  used  and 
followed  by  an  aqueous  solution  of  Bismarck-brown.  The  men- 
ingococci  will  be  brown. 


"jizsiiZz""     ....  . .... j 

Fig.  142. — Technic  of  spinal  puncture.  The  patient  is  sitting  on  the  edge 
of  a  chair  and  is  bent  forward;  the  crests  of  the  ilia  are  indicated  by  black 
lines,  and  are  on  a  level  with  the  spinous  process  of  the  fourth  lumbar  vertebra; 
the  "soft  spot"  is  found  just  above.  The  first  tube  receives  the  first  few  drops  of 
fluid,  which  are  usually  blood  tinged.  (Kolraer.) 

Cultivation. — The  organism  was  successfully  cultivated  by 
Weichselbaum,  but  does  not  readily  adapt  itself  to  artificial  media. 
It  develops  upon  agar-agar  and  glycerin  agar-agar,  upon  LofBer's 
blood-serum  mixture,  and,  according  to  Goldschmidt,t  upon  potato. 
Weichselbaum  did  not  find  that  it  developed  upon  potato.  It  does 
not  grow  in  bouillon  or  gelatin.  The  cultures  are  usually  scanty 
and  without  characteristic  features. 


"Jour.  Medical  Research,"  1906,  xiv,  89. 
"Centralbl.  f.  Bakt.  u.  Parasitenk.,"  n,  22,  23. 


390  Cerebro-spinal  Meningitis 

Flexner*  found  that  the  difficulties  of  cultivation  were  greatly 
reduced  by  the  employment  of  sheep-serum  instead  of  human 
serum.  Sheep-serum  water  was  prepared  according  to  the  method 
of  Hiss  (sheep-serum  i  part,  water  2  parts,  sterilized  in  the  auto- 
clave) and  mixed  with  a  beef-infusion  agar-agar  containing  2  per 
cent,  of  glucose.  The  quantity  of  sheep-serum  need  not  exceed  l^o 
to  J/10  of  the  volume  of  the  agar-agar  It  is  added  to  the  sterile 
melted  agar,  which  is  afterward  slanted  in  test-tubes  or  allowed  to 
congeal  on  the  expanded  surface  of  i6-ounce  Blake  bottles  when 
mass  cultures  are  to  be  used.  There  is  nothing  characteristic 
about  the  cultures.  The  cocci  grow  only  at  the  temperature  of 
the  body,  attain  only  a  sparse  development,  and  form  a  more  or 
less  confluent  line  of  minute,  rounded,  grayish  colonies  which  are 
easily  overlooked  upon  opaque  media  like  blood-serum.  The 
general  characteristics  of  the  growth  are  not  unlike  those  of  the 
pneumococcus,  streptococcus,  and  gonococcus. 

Colonies. — When  grown  upon  agar-agar  plates,  the  deep  colonies 
scarcely  develop  at  all,  appearing  under  the  low-power  lens  as 
minute,  irregularly  rounded,  granular  masses.  The  surface  colonies 
are  larger,  and  consist  of  an  opaque  yellowish-brown  nucleus  about 
which  a  flat,  rounded  disk  spreads  out.  The  edges  may  be  dentate; 
the  color  is  grayish  or  yellowish  near  the  center,  becoming  less 
intense  as  the  thin  edges  are  reached;  the  structure  is  finely  granular. 

Vital  Resistance. — The  vitality  of  the  culture  is  low,  and  the 
cocci  die  quickly.  It  becomes  necessary,  therefore,  when  studying 
the  organism  to  transplant  it  frequently — Parkf  says  every  two 
days.  FlexnerJ  found  that  they  do  not  survive  beyond  two  or 
three  days  and  that  transplantations  'do  not  succeed  unless  con- 
siderable quantities  of  the  culture  are  placed  upon  the  surface  of 
the  fresh  medium,  showing  that  many  of  the  organisms  were  already 
dead.  This  is  confirmed  by  the  microscopic  appearance  of  the 
cultures.  Those  sixteen  to  twenty-four  hours  old  stain  sharply 
and  uniformly;  on  the  second  day  many  of  the  cocci  show  irregularities 
of  size  and  staining,  and  after  several  days  no  normal-looking  cocci 
can  be  found.  It  was  found,  however,  that  in  carefully  preserved 
cultures  of  certain  strains  a  few  cocci  might  survive  for  many 
months.  Vitality  is  preserved  longest  when  the  cultures  are  kept 
in  the  thermostat  and  not  taken  out  when  grown,  to  be  kept  at  room 
temperature  or  in  a  refrigerator.  The  addition  of  a  small  quantity 
of  a  calcium  salt  favors  prolonged  vitality  and  will  sometimes  main- 
tain it  for  four  or  five  weeks  in  cultures  that  would  otherwise  die 
in  a  few  days.  Sodium  chlorid  is  injurious  to  the  cocci.  Flexner 
attributed  the  a-utolysis  of  the  cultures  to  an  enzyme. 

The  organism  is  soon  killed  by  drying,  by  exposure  to  the  sun, 

*  "Jour.  Experimental  Med.,"  1907,  ix,  p.  105. 

t  "Bacteriology  in  Medicine  and  Surgery,"  1899,  p.  362. 

j  Loc.  cit. 


Pathogenesis  391 

and  by  quite  moderate  variations  of  temperature.  It  succumbs  to 
very  high  dilutions  of  most  germicides  in  a  very  short  time. 

The  thermal  endurance  of  the  organism  is  very  slight.  It  will 
not  grow  except  at  37°C.,  ceases  to  grow  at  4o°C.  It  is  killed  in 
five  minutes  at  6o°C. 

Agglutination. — When  animals  are  immunized  by  repeated 
injections  of  the  Diplococcus  intracellularis,  their  blood-serum 
and  body-juices  become  agglutinative.  Such  serums  kept  in  the 
laboratory  can  be  used  for  the  identification  of  the  coccus  in  fresh 
culture,  though  the  reaction  is  not  exact,  since  the  agglutinability 
of  different  strains  of  cocci  is  different.  The  serums  have  an  ag- 
glutinating power  that  varies  from  i  :  500  to  i  :  3000  in  the  hands  of 
different  observers. 

Metabolic  Products. — The  meningococcus  breaks  up  dextrose  and 
maltose  with  the  production  of  acids,  but  has  no  similar  action  upon 
levulose,  saccharose,  or  inulin.  Acid  production  is  unaccompanied 
by  gas  evolution.  To  determine  the  acid  the  coccus  may  be  grown 
upon  acetic-fluid  agar  containing  the  sugar  under  examination,  and 
a  little  litmus  or  neutral  red. 

No  indol  is  produced,  no  gelatin-softening  coagulating  or  other 
ferments  are  formed. 

The  meningococcus  produces  an  endotoxin.  Albrech  and  Ghon* 
were  able  to  kill  white  mice  with  dead  cultures.  Lepierref  obtained 
a  toxin  from  bouillon  cultures  by  precipitating  them  with  alcohol. 

Pathogenesis. — The  results  of  animal  inoculations  made  with 
Diplococcus  intracellularis  meningitidis  are  disappointing.  Sub- 
cutaneous inoculations  into  the  lower  animals  are  continually  with- 
out effect.  Intrapleural  and  intraperitoneal  injections  of  cultures 
of  the  organism  into  mice  and  guinea-pigs  are  sometimes  fatal, 
the  dead  animals  showing  a  serofibrinous  inflammation  with  the 
presence  of  the  cocci.  The  intravenous  injection  of  the  coccus  into 
rabbits  is  followed  by  death  without  important  or  conclusive  symp- 
toms, and  usually  without  the  presence  of  cocci  in  the  blood. 

Weichselbaum  endeavored  to  reproduce  the  original  cerebro- 
spinal  meningitis  in  animals  by  trephining  and  injecting  the  cocci 
beneath  the  dura.  In  this  manner  he  inoculated  three  rabbits  and 
three  dogs.  Two  of  the  rabbit  injections  failed,  probably  because 
the  injected  material  escaped  at  once  from  the  wound.  The  third 
rabbit  died,  and  showed  marked  congestion  of  the  membranes  of 
the  brain  and  a  minute  softened  and  hemorrhagic  area.  In  these 
the  cocci  were  found  by  culture  to  be  abundant.  The  three  dogs  all 
died  with  congestion  and  pus-formation  in  the  membranes  and 
areas  of  softening  in  the  brain  substance.  The  cocci  were  recovered 
from  two  of  the  dogs,  but  the  lesions  of  the  third  animal,  which  lived 
twelve  days,  contained  none. 

*  "Wiener  klin.  Wochenschrift,"  1901. 

t  "Jour,  de  phys.  et  de  path,  gen.,"  v,  No.  3.  - 


392  Cerebrospinal  Meningitis 

Flexner*  found  that  in  large  doses  the  coccus  was  always  capable 
of  killing  small  guinea-pigs  and  mice  when  injected  intraperitoneally. 
To  achieve  this,  however,  the  organisms  should  be  suspended  in 
sheep-serum  water,  not  in  salt  solution,  which  is  an  active  poison  to 
them. 

Bettencourt  and  Francaf  tried  to  infect  monkeys  by  trephining, 
by  injecting  into  the  spinal  canal,  and  by  rubbing  the  cocci  upon 
the  nasal  mucous  membranes,  but  without  success.  Von  Lingel- 
sheim  and  LeuchsJ  and  Flexner§  were  more  successful.  Flexner's 
method  was  to  introduce  a  hypodermic  needle  into  the  spinal  canal, 
wait  until  a  few  drops  of  cerebro-spinal  fluid  had  escaped,  and  then 
inject  the  culture.  When  thus  introduced  at  a  low  level  of  the  spinal 
canal,  the  diplococci  distribute  themselves  through  the  meninges  in 
a  few  hours  and  excite  an  acute  meningitis,  the  exudate  of  which 
accumulates  chiefly  in  the  lower  spinal  meninges  and  the  meninges 
of  the  base  of  the  brain.  The  inflammation  extends,  in  monkeys, 
into  the  membranes  covering  the  olfactory  lobes  and  along  the 
dura  mater  into  the  ethmoid  plate  and  nasal  mucosa. 

The  nasal  mucous  membrane  is  found  in  many  instances  to  be 
inflamed  and  beset  with  hemorrhages.  Smear  preparations  from 
the  nasal  mucosa  show  many  polymorphonuclear  leukocytes  con- 
taining the  cocci  in  a  degenerated  form.  The  cocci  were  not  culti- 
vated from  the  nasal  exudates. 

Mode  of  Infection. — It  is  not  known  by  what  channels  infection 
with  Diplococcus  intracellularis  meningitidis  takes  place.  Weich- 
selbaum  supposed  it  might  enter  by  the  nasal,  auditory,  or  other 
passages,  especially  the  nose,  where  he  constantly  found  it,  and  the 
more  recent  studies  of  Goodwin  and  Sholly||  have  shown  the  organ- 
isms to  be  of  frequent  occurrence  in  the  nasal  cavities  of  meningitis 
patients  as  well  as  occasionally  in  those  associated  with  them.  It 
thus  becomes  evident  that  association  with  the  diseased  may  lead 
to  the  infection  of  the  well,  and  that  the  cases  should  be  isolated. 
The  same  conclusions  were  reached  by  Kolle  and  Wassermann,** 
who  studied  the  nasal  secretions  of  112  healthy  individuals,  not 
exposed  to  the  disease,  without  finding  any  cocci,  but  found  them 
in  the  nasopharynx  of  the  father  of  a  child  suffering  from  the  dis- 
ease, and  that  of  another  child  with  suspicious  symptoms. 

Steel 1 1  has  found  what  may  be  a  variety  of  the  meningococcus 
in  the  simple  posterior  basic  meningitis  of  infants.  The  organism 
differs  from  that  of  Weichselbaum  in  having  a  greater  longevity  upon 
culture-media,  where  it  often  lives  as  long  as  thirty  days.  It  is 

*  Loc.  cit. 

t  "Zeitschr.  f.  Hyg.  u.  Infekt.,"  XLVI,  p.  463. 

j  "Klin.  Jahrbuch,"  1906,  xv,  p.  489. 

§  Loc.  cit. 

||  "Journal  of  Infectious  Diseases,"  1906,  Supplement  No.  2,  p.  21. 

**  "Klinisches  Jahrbuch,"  xv,  1906. 

ft  "Pediatrics,"  Nov.  15,  1898. 


Specific  Therapy  393 

easily  stained  by  methylene  blue,  but  not  by  Gram's  method. 
Another  similar  organism  has  been  described  by  Elser  and  Huntoon.* 

Bacteriological  Diagnosis. — In  cases  with  the  clinical  symptoms 
of  meningitis,  the  bacteriological  diagnosis  is  of  great  assistance  in 
determining  the  correctness  of  the  diagnosis  and  the  nature  of  the 
infection.  It  is  accomplished  by  means  of  the  lumbar  puncture 
(vide  supra)  and  the  study  of  the  cerebro-spinal  fluid  thus  secured. 
Normal  cerebro-spinal  fluid  is  clear,  that  in  meningitis  is  cloudy.  A 
few  cubic  centimeters  of  the  fluid  can  be  used  for  culture  and  in- 
oculation experiments  of  as  many  kinds  as  are  deemed  advisable. 
The  remainder  is  placed  in  a  tube  and  whirled  in  a  centrifuge.  From 
the  sediment,  smears  are  made  upon  slides  and  stained  by  various 
methods,  including  Gram's  method.  If  the  chief  cells  appearing 
in  the  sediment  are  lymphocytes,  tuberculous  meningitis  should  be 
thought  of  and  smears  stained  for  tubercle  bacilli,  and  guinea-pigs 
inoculated.  If  the  cells  are  polymorphonuclear  cells,  tuberculous 
meningitis  is  usually  excluded.  If  small  cocci  are  found,  chiefly  in 
the  cells,  the  next  question  is  their  reaction  to  the  Gram  stain.  If 
positive  to  the  stain,  the  pneumococcus  should  be  thought  of;  if 
negative  to  the  stain,  the  meningococcus.  If  the  suspected  organ- 
ism grows  readily  upon  ordinary  culture-media,  it  is  not  the  meningo- 
coccus; if  it  grow  only  in  the  special  media  it  is  probably  the  men- 
ingococcus. Finally,  the  agglutinative  test  with  diluted  antiserum 
may  be  made  to  perfect  the  diagnosis. 

Specific  Therapy. — Kolle  and  Wassermannf  carefully  studied 
antimeningococcus  sera  for  specific  opsonins,  for  bacteriotropic 
substances,  and  for  other  evidences  of  favorable  therapeutic  action, 
but  came  to  no  definite  conclusions.  Flexner  and  JoblingJ  had 
better  success  both  in  developing  the  experimental  and  practical 
knowledge  of  the  serum.  The  serum  was  prepared  first  with  goats 
and  then  with  horses,  the  animals  being  injected  with  suspensions 
of  the  meningococci.  The  serum  is  used  by  injecting  it  into  the 
spinal  canal  through  a  lumbar  puncture.  The  precaution  must 
be  taken  to  permit  some  of  the  fluid  to  escape  first,  and  then  re- 
place it  by  the  antiserum,  of  which  not  more  than  30  cc.  must  be 
injected.  Several  such  injections  should  be  made.  Tabulations 
of  the  results  following  the  employment  of  Flexner's  serum  show  a 
large  percentage  of  recoveries. 

*  "Journal  of  Medical  Research,"  1909,  xx,  377. 

t  Loc.  cit. 

J  "Jour.  Experimental  Medicine,"  1907,  ix,  p.  168,  and  1908,  x,  p.  141. 


CHAPTER  VIII 
GONORRHEA 

MICROCOCCUS  GONORRHOEA  (NEISSER) 

General  Characteristics. — A  minute,  biscuit-shaped,  non-motile,  non-sporo 
genous,  non-liquefying,  non-chromogenic,  non-flagellate,  aerobic,  strictly  para 
sitic  coccus,  not  stained  by  Gram's  method,  cultivable  upon  special  media,  an< 
pathogenic  for  man  only. 

All  authorities  now  accept  the  "gonococcus"  as  the  specifi 
cause  of  gonorrhea.  It  was  first  observed  in  the  urethral  and  con 
junctival  secretions  of  gonorrhea  and  purulent  ophthalmia  b; 
Neisser*  in  1879. 

Bummf  found  other  cocci  closely  resembling  the  gonococcu 
in  the  inflamed  urethra,  and  points  out  that  neither  its  shape  no 
its  position  in  the  cells  can  be  regarded  as  characteristic,  but  tha 
failure  to  stain  by  Gram's  method  can  alone  enable  us  to  say  wit] 
certainty  that  biscuit-shaped  cocci  found  in  urethral  pus  ar 
gonococci. 

Distribution. — The  gonococcus  is  a  purely  parasitic  pathogeni 
organism.  It  can  be  found  in  the  urethral  discharges  of  gonorrhe; 
from  the  beginning  until  the  end  of  the  disease,  and  often  for  man; 
months  and  even  years  after  recovery  from  it.  After  the  perioc 
of  creamy  pus  has  passed,  its  numbers  are  usually  outweighed  b] 
other  pyogenic  organisms.  WertheimJ  cultivated  the  gonococcu 
from  a  case  of  chronic  urethritis  of  two  years'  standing,  and  prove< 
its  virulence  by  producing,  experimental  gonorrhea  in  a  humai 
being.  The  organisms  are  chiefly  found  within  the  pus-cells  o 
attached  to  the  surface  of  epithelial  cells,  and  should  always  b< 
sought  for  as  diagnostic  of  gonorrhea,  as  purulent  urethritis  is  some 
times  caused  by  other  organisms,  as  Bacillus  coli  communis§  am 
Staphylococcus  pyogenes. 

Morphology. — The  organisms  occur  in  pairs.  Each  pair  of  younj 
cocci  is  composed  of  two  spherical  organisms,  but  as  they  grow  olde 
the  inner  surfaces  become  flattened  and  separated  from  one  anothe 
by  a  narrow  interval,  so  that  they  somewhat  resemble  a  coffee 
bean.  A  pair  of  the  cocci  resembles  the  German  biscuit,  and  i 
described  by  the  Germans  as  semmelformig. 

*  "Centralbl.  f.  d.  med.  Wissenschaft,"  1879,  No.  28. 

t  "Der  Mikroorganismus  der  gonorrhoischen  Schleimhauterkrankungen,' 
"Gonococcus  Neisser,"  second  edition,  1887.  , 

J  "  Archiv  f.  Gynakologie,"  1892,  Bd.  XLII,  Heft  i. 

§  Van  der  Pluyn  and  Loag,  "  Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Feb.  28,  1895 
Bd.  xyn,  Nos.  7,  8,  p.  233. 

394 


Isolation  and  Cultivation  395 

The  gonococci  are  small,  the  length  of  one  of  the  coffee-bean  cocci 
being  about  1.6  ju,  its  breadth  about  0.8  /JL.  They  are  not  motile, 
nor  provided  with  flagella,  and  are  without  spores. 

Quite  as  characteristic  as  the  form  of  the  organism  is  its  rela- 
tion to  the  cells.  In  most  of  the  inflammatory  exudates  the  gono- 
cocci are  contained  either  in  epithelial  cells  or  in  leukocytes,  very 
few  of  them  lying  free.  This  intracellular  position  is  supposed  to 
depend  upon  active  phagocytosis  of  the  cocci  by  the  cells.  It  may 
not  obtain  in  old  lesions. 

Staining. — They  stain  readily  with  all  the  aqueous  solutions  of 
the  anilin  dyes — best  with  rather  weak  solutions,  but  not  by  Gram's 
method. 


Fig.  143. — Gonococci  in  urethral  pus. 

The  organisms  contained  in  pus  can  be  beautifully  shown  by 
first  treating  the  prepared  film  with  alcoholic  eosin,  and  then  with 
Loffler's  alkaline  methylene  blue.  A  differential  color  test  can  be 
made  by  staining  the  film  by  Gram's  method  and  then  with  aqueous 
Bismarck  brown,  or,  what  may  be  still  better,  with  3  per  cent, 
aqueous  solution  of  pyronin.  Ordinary  pus  cocci,  taking  the 
Gram's  stain,  appear  blue-black;  the  gonococci,  taking  the  counter- 
stain,  are  brown  in  the  former,  purplish  red  in  the  latter  case. 

Isolation  and  Cultivation. — The  organism  does  not  grow  upon 
any  of  the  ordinary  culture-media,  and  grows  very  scantily  upon 
any  artificial  medium.  Wertheim*  succeeded  in  cultivating  it  by- 
diluting  a  drop  of  gonorrheal  pus  with  human  blood-serum,  mixing 
this  with  an  equal  part  of  melted  2  per  cent,  agar-agar  at  4o°C., 
and  pouring  the  mixture  into  Petri  dishes,  which,  as  soon  as  the 
medium  became  firm,  were  stood  in  the  incubator  at  37°C.  or, 
preferably,  4o°C.  In  twenty-four  hours  the  colonies  could  be 
observed.  Those  upon  the  surface  showed  a  dark  center,  sur- 
rounded by  a  delicate  granular  zone. 

*  "Archiv.  fur  Gynakologie,"  1892. 


396  Gonorrhea 

Young*  had  excellent  success  with  a  hydrocele-agar  prepared  as 
follows: 

"The  fluid  (hydrocele  or  ascitic)  is  obtained  sterile,  the  locality  of  the  puncture 
being  carefully  sterilized  by  modern  surgical  methods,  the  sterile  trocar  covered 
at  its  external  end  with  sterilized  gauze  so  as  not  to  be  infected  by  the  operator's 
hand,  and  the  fluid  collected  in  sterile  flasks,  the  sterile  stoppers  being  then  re- 
placed. Collecting  the  fluid  in  this  way  we  have  very  rarely  had  it  contaminated, 
often  keeping  it  several  months  before  using  it.  The  fluid  is  mixed  with  ordi- 
nary nutrient  agar.  A  number  of  common  slants  are  put  in  the  autoclave  for 
five  minutes.  This  liquefies  the  agar  and  at  the  same  time  thoroughly  sterilizes 
the  tubes  and  cotton  stoppers.  The  slants  are  then  put  in  a  water-bath  at  55°C. 
so  as  not  to  coagulate  the  albumin  when  mixed  with  the  agar.  The  stopper  hav- 
ing been  removed  from  a  small  flask  of  hydrocele  fluid,  the  top  of  the  flask  is 
flamed  and  the  albuminous  fluid  is  then  poured  into  an  agar  tube  (the  top  of 
which  has  also  been  flamed)  in  proportions  a  little  more  than  one  to  two."  The 
medium  can  be  allowed  to  solidify  in  tubes  or  can  be  poured  into  Petri  dishes. 

When  one  of  the  colonies  was  transferred  to  a  tube  of  human 
blood-serum,  or  of  one  of  the  above-described  mixtures  obliquely 
coagulated,  isolated  little  gray  colonies  occur,  later  becoming  con- 
fluent and  producing  a  delicate  smeary  layer  upon  the  medium. 
The  main  growth  is  surrounded  by  a  thin,  veil-like  extension  which 
gradually  fades  away  at  the  edges.  A  slight  growth  occurs  in  the 
water  of  condensation. 

Heimanf  found  that  the  gonococcus  grows  best  in  a  mixture  of 
i  part  of  pleuritic  fluid  and  2  parts  of  2  per  cent.  agar.  WrightJ 
prefers  a  mixture  of  urine,  blood-serum,  peptone,  and  agar-agar. 

Wassermann§  used  a  mixture  of  15  cc.  of  pig-serum,  35  cc.  of 
water,  3  cc.  of  glycerin,  and  2  per  cent,  of  nutrose.  The  nutrose  is 
dissolved  by  boiling  and  the  solution  sterilized.  This  is  then  added 
to  agar,  in  equal  parts,  and  used  in  plates.  || 

Laitinen**  found  agar-agar  mixed  with  one-third  to  one-half  its 
volume  of  cyst  or  ascitic  fluid,  and  bouillon  containing  i  per  cent, 
of  peptone  and  0.5  per  cent,  of  sodium  chlorid,  mixed  with  one- 
third  to  one-half  its  volume  of  cyst  or  ascitic  fluid,  very  satisfactory. 
The  gonococcus  could  be  kept  alive  upon  these  media  for  two  months. 
Laitinen  found  that  the  gonococcus  produces  acids  in  the  early 
days  of  its  development,  and  alkalies  subsequently.  He  was  unable 
to  isolate  any  toxin  from  the  cultures. 

Vital  Resistance. — Authorities  agree  that  the  gonococcus  has  very 
slight  power  of  heat  endurance.  Wertheim  found  the  optimum 
temperature  of  cultivation  to  be  39°  to  4o°C.,  and  saw  no  harm 
result  from  exposure  to  42°C.  It  is  killed  in  a  few  minutes  at  55°C. 
The  gonococci,  though  not  easily  cultivated,  are  said  to  resist 
unfavorable  conditions,  especially  drying,  very  well.  Kratter  was 

*  "  Contributions  to  the  Science  of  Medicine  by  the  Pupils  of  William  M. 
Welch,"  Baltimore,  1900,  p.  677. 

t  'Medical  Record,"  Dec.  19,  1886. 
J  'Amer.  Jour.  Med.  Sci.,"  Feb.,  1895. 
§  'Berliner  klin.  Wochenschrift,"  1897. 

||    'See  "Text-Book  of  Bacteriology,"  by  Hiss  and  Zinsser,  1910,  p.  383. 
**  'Centralbl.  f.  Bakt.  u.  Parasitenk.,"  June  i,  1898,  vol.  xn,  No.  20,  p.  874. 


Toxic  Products  397 

able  to  demonstrate  their  presence  upon  washed  clothing  after  six 
months,  and  found  that  they  still  stained  well.  This  may  not  mean 
that  the  organisms  were  still  alive. 

In  artificial  culture  the  gonococcus  soon  dies,  though  cultures 
from  different  sources  differ  considerably  in  this  regard.  As  a 
rule  they  survive  but  a  few  transplantations,  though  Young  found 
that  one  culture  had  been  kept  alive  by  students  in  his  laboratory 
for  more  than  three  months. 

Diagnosis. — The  diagnosis  of  gonorrhea  by  finding  the  diplococci 
in  urethral  pus  and  epithelial  cells  is  a  very  simple  matter.  The 
recognition  of  the  micro-organisms  under  other  conditions  is  by 
no  means  easy.  Thus,  when  gonorrhea  becomes  chronic  and  the 
cocci  are  no  longer  taken  up  by  the  phagocytes,  it  raises  a  little 
doubt  whether  Gram-negative  cocci  may  be  true  gonococci  or  not, 
yet  it  is  at  precisely  this  time  when  a  patient  getting  over  gleet  and 
wanting  to  marry  desires  to  know  definitely  whether  gonococci  are 
any  longer  present  in  his  urethra  or  not.  Again,  when  the  gonococ- 
cus-like  organisms  occur  upon  the  conjunctiva,  in  the  pus  taken  from 
joints,  upon  the  valves  of  the  heart,  or  in  the  Fallopian  tubes,  the 
same  .difficulty  is  met.  Probably  the  greatest  perplexity  arises 
when  the  conjunctiva  is  called  in  question,  for  here  there  can  come 
about  a  confusion  of  the  gonococcus,  the  pneumococcus,  and  Micro- 
coccus  catarrhalis  (q.v.)  which  only  careful  staining  and  culture  ex- 
periments can  solve.  The  pneumococcus  may  be  readily  separated 
if  its  lanceolate  form  and  capsules  can  be  observed,  but  it  is  only 
by  seeing  that  Micrococcus  catarrhalis  grows  readily  and  luxuriantly 
upon  all  the  laboratory  media,  and  the  gonococcus  with  difficulty 
and  very  sparingly  upon  any  media,  that  the  diagnosis  can  be  made 
with  certainty. 

The  method  of  diagnosis  by  staining  and  looking  for  Gram- 
negative  diplococci  in  the  cells  is  only  a  " rough  and  ready"  one 
and  is  not  dependable. 

The  method  of  complement  fixation  is  probably  the  court  of  final 
resort,  but  this  test  is  attended  with  considerable  technical  difficulty. 

Toxic  Products. — The  toxic  metabolic  products  of  the  gonococcus 
appear  to  be  contained  within  the  bodies  of  the  bacteria  and  dis- 
seminated but  slightly  throughout  the  culture-media.  Christmas,* 
Nicolaysen,f  and  Wassermann  J  have  studied  gonotoxin,  and  have  all 
found  that  it  remains  in  the  bodies  of  the  bacteria.  The  toxin 
seems  to  be  quite  stable  and  is  not  destroyed  by  temperatures  fatal 
to  the  cocci.  Wassermann  obtained  some  cultures  of  which  o.i 
cc.  would  kill  mice;  others,  of  which  i.o  cc.  was  required.  The 
poison  can  be  precipitated  with  absolute  alcohol.  Small  quantities 
of  the  toxin  introduced  into  the  urethra  cause  suppuration  at  the 

*  "Ann.  de  1'Inst.  Pasteur,"  1897. 

t  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1897,  Bd.  xxn,  Nos.  12  and  13,  p.  305. 
t  "Zeitschrift  fur  Hygiene,"  1898,  and  "Berliner  klin.  Wochenschrift,"  1897, 
No.  32,  p.  685. 


398  Gonorrhea 

point  of  application,  fever,  swelling  of  the  neighboring  lymphatic 
nodes,  and  muscular  and  articular  pains. 

Pathogenesis. — It  is  generally  believed  that  gonorrhea  cannot 
be  communicated  to  animals. 

There  is  no  doubt  but  that  the  gonococcus  causes  gonorrhea, 
as  it  has  on  several  occasions  been  intentionally  and  experimentally 
inoculated  into  the  human  urethra  with  resulting  typical  disease. 
It  is  constantly  present  in  the  disease,  and  very  frequently  in  its 
sequelae,  though  it  not  infrequently  happens  that  the  lesions  second- 
ary to  gonorrhea  are  caused  by  the  more  common  organisms  of 
suppuration  that  have  entered  through  the  surface  denudations 
caused  by  the  gonococcus. 

Opinions  differ  as  to  whether  the  gonococci  can,  with  equal  facility, 
penetrate  squamous  and  columnar  epithelium.  Their  attacks  are 
usually  made  upon  surfaces  covered  with  squamous  epithelium. 

The  injection  of  gonococci  into  the  subcutaneous  tissue  is  not 
followed  by  either  abscess  formation  or  septic  infection. 

Gonococci  rarely  enter  the  circulation  of  human  beings  and  occa- 
sion a  peculiar  septic  condition  with  irregular  temperature,  apt  to 
be  followed  by  invasion  of  the  cardiac  valves,  joints,  or  other 
tissues.  P.  Kraus*  has  twice  succeeded  in  cultivating  the 
gonococcus  from  the  blood  of  patients  in  the  stage  of  septic 
infection. 

The  deep  lesions  caused  by  the  gonococcus  are,  however,  numer- 
ous, and  in  Young's  paper  (loc.  cit.}  its  widespread  powers  of  pyo- 
genic  infection  are  well  shown  in  a  collection  of  the  cases  recorded 
in  the  literature,  and  some  original  observations  showing  the  un- 
doubted occurrence  of  the  gonococcus  in  gonorrhea,  ophthalmia 
neonatorum,  arthritis,  tendosynovitis,  perichondritis,  subcutaneous 
abscess,  intramuscular  abscess,  salpingitis,  pelvic  peritonitis,  adenitis, 
pleuritis,  endocarditis,  septicemia,  acute  cystitis,  chronic  cystitis, 
pyonephrosis,  and  diffuse  peritonitis. 

In  the  beginning  of  the  inflammatory  process  the  cocci  grow 
in  the  superficial  epithelial  cells,  but  soon  penetrate  between  the 
cells  to  the  deeper  layers,  where  they  continue  to  keep  up  the  irri- 
tation as  the  superficial  cells  desquamate. 

All  urethral  inflammations,  and  in  gonorrhea  all  of  the  inflam- 
matory symptoms,  do  not  depend  upon  the  gonococcus.  The 
periurethral  abscess,  salpingitis,  etc.,  not  infrequently  depend  upon 
ordinary  pus  cocci,  and  the  author  has  seen  a  case  of  gonorrhea  with 
double  orchitis,  general  septic  infection,  and  endocarditis  in  which 
the  gonococci  had  no  role  in  the  sepsis,  which  was  caused  by  a  large 
coccus  that  stained  beautifully  by  Gram's  method. 

In  the  remote  secondary  inflammations  the  gonococci  disappear 
after  a  time,  and  the  inflammation  either  subsides  or  is  maintained 
~*  "Berliner  klin.  Wochenschrift,"  May  9,  1904,  No.  19,  p.  494- 


Immunization  399 

by  other  bacteria.  In  synovitis,  however,  the  inflammation  excited 
may  last  for  months. 

So  long  as  the  gonococci  persist  in  his  urethra  or  other  superficial 
tissues  the  patient  may  spread  the  contagion,  and  after  apparent 
recovery  from  gonorrhea  the  cocci  may  remain  latent  in  the  urethra 
for  years,  not  infrequently  causing  a  relapse  if  the  patient  partake 
of  some  substance,  as  alcohol,  irritating  to  the  mucous  membranes. 
Bearing  this  in  mind,  physicians  should  be  careful  that  their  patients 
are  not  too  soon  discharged  as  cured  and  permitted  to  marry. 

Immunization  against  the  gonococcus  has  not  yet  been  success- 
fully achieved.  Wassermann  failed  altogether;  Christmas  claims 
to  have  immunized  goats,  but  the  serum  of  these  animals  could 
not  be  shown  to  contain  any  antitoxin  or  to  be  bacteriolytic. 

Torrey*  prepared  an  antigonococcus  serum  by  immunizing 
rabbits  with  gonotoxin.  The  culture  used  was  isolated  from  a 
case  of  acute  gonorrhea  in  a  medium  of  rich  ascitic  fluid  and  slightly 
acid  beef  infusion,  peptone  broth,  equal  parts.  In  speaking  about 
this  mixture  Dr.  Torrey  said  that  the  exact  reaction  was  its  most 
important  feature,  as  otherwise  the  gonococci  soon  died.  Tubes  of 
about  12  cm.  of  the  mixture  were  heated  to  about  6o°C.  for  several 
hours  and  then  tested  for  sterility.  The  cocci  were  cultivated  at 
36°  to  37°C.  After  eighteen  to  twenty-four  hours'  incubation  a 
slight  granular  growth  appears  near  the  surface  and  on  the  sides  of 
the  tube.  This  slowly  increases  until  after  six  days  the  medium  is 
well  clouded  on  shaking.  Large  rabbits  were  used  for  making  the 
serum,  and  were  intraperitoneally  inoculated  with  10  cc.  of  an 
entire  culture.  The  first  inoculation  resulted  in  a  loss  of  weight, 
sometimes  amounting  to  one-fourth  of  the  body-weight.  After  an 
interval  of  five  or  six  days  a  second  injection  is  given,  then  after  a 
similar  interval,  a  third,  and  so  on.  The  best  results  were  ob- 
tained when  cultures  from  six  to  fifteen  days  old  were  employed. 
The  rabbits  were  bled  for  the  first  time  after  the  sixth  dose,  as  if 
the  treatment  be  pushed  they  soon  fall  into  a  state  of  cachexia, 
rapidly  emaciate,  and  die.  Each  animal  furnishes  70  to  90  cm. 
of  the  serum,  which  was  inclosed  in  2-cm.  bulbs,  hermetically  sealed, 
and  kept  without  any  preservative. 

With  serum  made  in  this  way  by  Torrey,  Rogersf  treated  a 
number  of  obstinate  cases  of  gonorrheal  rheumatism,  with  appar- 
ently good  results. 

Good  results  in  gonorrheal  arthritis  and  in  gleet  are  also  claimed 
for  treatment  with  gonococco-vaccines. 

*  "Journal  Amer.  Med.  Assoc.,"  Jan.  27,  1906,  XLVI,  p.  261. 
t  "Jour.  Amer.  Med.  Assoc.,  Jan.  27,  1609,  "XLVI,  .p.  261. 


CHAPTER  IX 
CATARRHAL  INFLAMMATION 

MICROCOCCUS  CATARRHALIS  (SEIFERT) 

General  Characteristics. — A  small,  slightly  ovoid,  non-motile,  non-sporulatin^ 
non-flagellated,  non-liquefying  aerobic  and  optionally  anaerobic,  non-chrome 
genie  coccus,  pathogenic  for  man,  and  not  for  the  lower  animals,  cultivable  upo 
the  ordinary  media,  staining  by  the  ordinary  methods,  but  not  by  Gram's  methoc 

This  micro-organism,  which  seems  to  be  closely  related  to  th 
staphylococci,  was  first  observed,  in  sections  of  the  lung  of  a  case  c 
influenza,  by  Seifert.*  It  was  successfully  cultivated  in  1890  b; 
Kirchnerf  from  10  cases  of  an  influenza-like  affection.  It  has  sine 
been  frequently  demonstrated  in  the  exudates  from  various  in 


Fig.  144. — Micrococcus  catarrhalis  in  smear  from  sputum  (F.  T.  Lord;  photo  b 

L.  S.  Brown). 

flammatory  conditions  of  the  respiratory  tract  and  conjunctive 
and  seems  to  be  a  not  uncommon  organism  of  superficial  inflamma 
tions.  It  is  a  rather  troublesome  organism,  causing  some  confusio: 
because  of  its  disposition  to  occur  in  pairs,  which  gives  it  a  clos 
resemblance  to  the  pneumococcus  except  in  cases  in  which  the  cap 

*  "  Volkmann's  klin.  Vortr.,"  Nr.  240. 
f'Zeitschr.  f.  Hyg.,"  Bd.  9. 
400 


Pathogenesis 


401 


sules  of  the  latter  are  distinct.  It  is  also  readily  taken  up  by  the 
leukocytes,  and  may  so  resemble  the  gonococcus;  and  it  is  not  al- 
ways easy,  perhaps  not  always  possible,  to  distinguish  it  from  the 
Diplococcus  intracellularis  meningitidis. 

Morphology. — The  organism  is  spheric  or  slightly  ovoid,  may 
occur  singly,  though  usually  appears  in  pairs  or  clusters.  Large 
numbers  are  enclosed  in  the  leukocytes  or  other  cells.  The  spheric 
organisms  have  a  diameter  of  about  i  M;  the  ovoid  organisms  may 
measure  as  much  as  1.5  by  2  ju.  The  relation  of  the  cocci  to  the 
cells  seems  to  have  something  to  do  with  the  course  of  the  inflam- 
matory conditions  with  which  they  are  asso- 
cia  ted.  D  uring  the  activity  of  the  process  large 
numbers  of  the  cocci  may  be  free;  toward  its 
close  they  may  all  be  enclosed  in  the  leukocytes. 

The  organisms  are  not  motile  and  they  have 
no  flagella. 

Staining. — The  cocci  stain  by  ordinary 
methods,  but  not  by  Gram's  method. 

Cultivation. — The  organism  can  be  easily 
cultivated,  and  thus  differentiates  itself  from 
the  fastidious  gonococcus.  The  colonies  are 
large,  white,  irregular  in  outline,  elevated  at 
the  center,  not  viscid,  and  grow  readily  at 
room  temperatures  upon  all  the  culture  media, 
the  best  upon  blood  agar-agar.  The  vitality 
of  the  organism  in  culture  is  not  great.  Very 
often  transplantation  made  after  from  four  to 
six  days  fail  to  grow;  and  in  the  cultures  one 
usually  finds  many  deeply  staining,  supposedly 
living  cocci,  and  many  poorly  staining,  sup- 
posedly dead  organisms. 

Agar-agar. — The  culture  in  general  re- 
sembles that  of  Staphylococcus  albus.  When 
blood  is  added  to  the  agar-agar,  the  growth 
is  more  luxuriant,  whitish,  and  usually  consists 
of  closely  approximated  colonies  which  do  not 
become  confluent. 

Gelatin. — This  medium  is  not  liquefied. 

Bouillon. — At  the  end  of  the  first  day  no  growth  seems  to  have 
taken  place,  but  at  the  end  of  the  second  day  there  is  a  slight  cloud- 
ing and  a  meager  precipitate.  The  organism  seems  to  maintain 
its  vitality  somewhat  longer  in  bouillon  than  in  other  culture- 
media. 

Pathogenesis. — The  organism  seems  to  be  scarcely  pathogenic 
for  animals.  Kirchner  was  able  to  kill  a  guinea-pig  by  intrapleural 
injection,  and  Neisser,  who  performed  numerous  experiments  upon 
mice,  guinea-pigs,  and  rabbits,  only  once  succeeded  in  producing  a 


Fig.  145. — Micrococ- 
cus  catarrhalis  colonies 
on  agar  (F.  T.  Lord; 
photo  by  L.  S.  Brown). 


4O2  Catarrhal  Inflammation 

fatal  infection,  by  the  intraperitioneal  injection  of  0.4  cc.  of  bouillon 
culture.  In  this  animal  the  cocci  were  found  in  all  the  internal 
organs.  As  has  already  been  said,  the  organism  is  found  associated 
with  superficial  inflammatory  conditions  of  the  mucous  membrane. 
It  is  probably  most  common  in  influenza.  It  has  also  been 
found  in  conjunctivitis,  in  bronchitis,  in  whooping-cough,  and  in 
pneumonia. 


CHAPTER  X 
CHANCROID 

THE  BACILLUS  DUCREYI 

General  Characteristics. — A  small,  ovoid  streptobacillus,  with  rounded,  deeply 
staining  ends,  non-motile,  non-flagellate,  non-sporogenous;  aerobic  and  optionally 
anaerobic,  non-chromogenic,  staining  by  ordinary  methods,  but  not  by  Gram's 
method,  cultivable  on  special  media  only  and  pathogenic  only  for  man  and  certain 
monkeys. 

The  chancroid,  soft  chancre,  or  non-specific  sore,  as  it  is  called, 
is  a  common  venereal  affection  of  both  sexes,  most  frequent  among 
those  who  give  little  attention  to  cleanliness.  It  is  characterized 
by  the  appearance  of  a  soft  reddish  papule,  which  makes  its  appear- 
ance usually  upon  the  genital  organs,  rarely  upon  other  parts  of  the 
body,  soon  after  the  infection,  and  soon  becomes  transformed  to  an 
ugly  ulceration  whose  usual  tendency  is  toward  slow  and  persistent 
enlargement,  though  in  different  cases  it  may  be  indolent,  active, 
phagedenic,  or  serpiginous.  The  inguinal  or  other  nearby  lymph- 
nodes  early  enlarge  and  soon  soften  and  ulcerate.  The  disease  is, 
therefore,  extremely  destructive  to  the  tissues  invaded,  though  no 
constitutional  involvement  ever  takes  place. 

Specific  Organism. — In  1889  Ducrey*  described  a  peculiar  organ- 
ism whose  presence  he  was  able  to  demonstrate  with  great  con- 
stancy, sometimes  in  pure  culture,  in  the  lesions  of  chancroid,  and 
which  he  believed  to  be  the  specific  organism  of  the  affection.  Unnaf 
later  described  an  organism  resembling  that  of  Ducrey,  and  the  later 
observations  of  Krefting,{  Peterson,§  Nicolle,||  Cheinisse,**  and 
Davisfj  have  abundantly  confirmed  the  observations  of  Ducrey  and 
Unna,  and  proved  the  identity  of  the  two  micro-organisms  and 
their  specificity  for  the  disease. 

Morphology. — The  organism  is  commonly  described  as  a  "  strepto- 
bacillus." It  is  very  small,  short,  and  ovoid  in  shape,  and  occurs 
habitually  in  longer  or  shorter  chains.  Each  organism  measures 
about  1.5  X  0.5  M.  The  ends  are  rounded  and  stain  deeply.  In 
pure  cultures  long  undivided  filaments,  at  least  twenty  times  as 
long  as  the  individual  bacilli,  are  not  uncommon.  There  seems  to  be 

*"Congres.  Inter,  de  Dermatol.  et  de  Syphilog.,"  Paris,  1889;  "Compt. 
rendu,"  p.  229. 

t  "Monatschr.  f.  praktische  Dermatologie,"  1892,  Bd.  xiv,  p.  485. 

j  "  Archiv.  f.  Dermatol.  u.  Syphilol.,"  1897,  p.  263;  1897,  p.  41. 

§  "Centralbl.  f.  Bakt.,"  etc.,  1893,  xm,  p.  743. 

||  "Med.  Moderne,"  Paris,  1893,  rv,  p.  735. 

'  "Ann.  de  Dermat.  et  de  Syphil.,"  Par.,  1894,  p.  272. 
ft  "Jour.  Med.  Research,"  1893,  ix,  p.  401. 

403 


404  Chancroid 

no  relation  between  the  cells  and  the  bacilli.  As  a  rule,  they  are 
free,  sometimes  they  are  inclosed  in  leukocytes.  The  bacilli  are  not 
motile,  have  no  flagella  and  do  not  form  spores. 

Staining. — The  organisms  are  somewhat  difficult  to  stain,  as  they 
do  not  retain  the  color  well,  giving  it  up  quickly  when  washed. 
They  do  not  stain  by  Gram's  method. 

Cultivation. — The  first  successful  isolation  and  cultivation  of  the 
organism  seems  to  have  been  by  Benzancon,  Griffon  and  Le  Sours* 
upon  a  culture-medium  consisting  of  rabbits'  blood  i  part,  and  agar- 
agar  2  parts.  Davis  f  has  been  equally  successful  in  cultivating  the 
organism  upon  this  medium.  His  method  was  as  follows: 

"Tubes  of  2  per  cent,  agar,  reaction  +1.5,  were  melted  and 
mixed  with  fresh  rabbits'  blood  drawn  under  aseptic  precautions, 


Fig.  146. — Smear  of  pus  of  chancroid  of  penis  stained  with  carbol-fuchsin 
and  briefly  decolorized  by  alcohol.  X  1500  (Davis).  (Photomicrograph  by 
Mr.  L.  S.  Brown.) 

in  the  proportion  of  two-thirds  agar  to  one-third  blood,  and  slanted 
while  in  a  fluid  state.  At  a  later  period  tubes  of  rabbits'  blood-serum 
uncoagulated,  also  rabbits'  blood  bouillon,  one-third  blood  to  two- 
thirds  bouillon,  were  used,  and  gave  equally  satisfactory  results. 
By  employing  small  tubes  of  freshly  drawn  human  blood  pure  cul- 
tures were  obtained  in  several  instances  from  genital  lesions,  direct, 
without  any  special  cleansing  of  the  ulcerated  surface.  This,  I 
believe,  is  the  best  medium  for  obtaining  cultures  from  a  source  open 
to  contamination,  the  fresh  blood  apparently  inhibiting  to  a  certain 
extent  the  growth  of  extraneous  organisms." 

No  growth  takes  place  upon  ordinary  culture-media  under  either 
aerobic  or  anaerobic  conditions. 

Cultures  are  best  obtained  by  puncturing  an  unopened  bubo  with 
a  sterile  needle  and  planting  the  pus  directly  and  immediately  upon 
the  special  medium  which  should  have  been  warmed  in  the  incubator 

*"Ann.  de  Dermat.  et  de  Syphilog.,"  1901,  n,  p.  i. 
t  Loc.  cit. 


Pathogenesis  405 

so  that  the  pus  is  not  chilled.     In  this  way  pure  cultures  which  are 
difficult  to  get  from  the  soft  sore  itself,  may  be  secured. 

Colonies. — The  colonies  appear  upon  the  appropriate  media  in 
about  twenty-four  hours,  and  attain  their  complete  development  in 
about  forty-eight  hours.  They  are  at  first  round  bright  globules, 
and  later  become  grayish  and  opaque.  They  measure  i  to  2  mm. 
in  diameter  and  never  become  confluent.  They  are  difficult  to  pick 
up  with  the  platinum  wire,  tending  to  slide  over  the  smooth  surface 
of  the  medium. 


r 

Fig.  147. — Culture  from  ulceration  on  monkey  resulting  from  inoculation  of 
culture  from  a  case  of  chancroid  of  finger,  first  generation.  Stained  with  carbol- 
f uchsin  and  briefly  decolorized  by  alcohol.  Culture  of  twenty-four  hours' 
growth  in  rabbit's  bouillon.  X  1500  (Davis).  (Photomicrograph  by  Mr.  L.  S. 
Brown.) 

Vital  Resistance. — The  organisms  seem  to  possess  little  vitality, 
their  life  in  artificial  culture  being  limited  to  a  few  days.  Fre- 
quent transplantation  enabled  Davis  to  carry  them  on  to  the 
eleventh  cultural  generation. 

Pathogenesis.— The  organism  is  pathogenic  for  man  and  certain 
monkeys  (macacus),  but  not  for  the  ordinary  laboratory  animals. 
The  organism  can  be  found  in  large  numbers  in  both  the  genital  and 
extragenital  chancroidal  lesions,  and  usually  in  small  numbers  in 
the  pus  from  chancroidal  buboes.  It  has  not  been  encountered 
elsewhere.  Lenglet*  isolated  the  organism  in  pure  culture,  and  by 
inoculation  with  his  cultures  reproduced  the  lesions  in  man. 

*"Bull.Med."  1898, p.  1051;  "Ann.  deDermatol.etdeSyph.,"  1901,11,  p.  209. 


CHAPTER  XI 
ACUTE  CONTAGIOUS  CONJUNCTIVITIS 

THE  KOCH- WEEKS  BACILLUS 

General  Characteristics. — A  minute,  slender  bacillus,  non-motile,  non-flagel- 
lated, non-sporogenous,  non-liquefying,  non-chromogenic,  aerobic,  and  optionally 
anaerobic,  staining  by  the  ordinary  methods  but  not  by  Gram's  method,  sus- 
ceptible of  cultivation  upon  special  media  only,  and  specific  for  acute  contagious 
conjunctivitis. 

Acute  contagious  conjunctivitis  is  a  common  and  world-wide 
affection,  sometimes  called  "pink  eye,"  and  sometimes  erroneously 
called  catarrhal  conjunctivitis.  All  its  characteristics,  and  es- 
pecially its  contagiousness,  point  to  its  being  a  specific  disease  due 
to  a  specific  cause,  and  thus  entirely  different  from  ordinary  non- 
specific catarrh. 

Specific  Micro-organism. — The  first  bacteriologic  investigation 
of  acute  contagious  conjunctivitis  was  made  by  Robert  Koch,* 
when  in  Egypt  investigating  a  cholera  epidemic.  While  in  Alex- 
andria he  examined  the  secretions  from  50  cases  of  conjunctivitis, 
finding  the  gonococcus,  or  an  organism  closely  resembling  it.  In  a 
less  severe  form  of  the  disease,  however,  he  found  a  peculiar  small 
bacillus.  He  seemed  satisfied  with  this  observation,  or  had  no  time 
to  pursue  the  matter  farther,  for  no  cultivation  or  other  experiments 
are  mentioned. 

The  organism  was  observed  from  time  to  time,  but  no  serious 
consideration  seems  to  have  been  devoted  to  it  until  Weeks  f  pub- 
lished an  account  of  what  seemed  to  be  the  identical  organism,  which 
he  not  only  observed,  but  also  cultivated,  and  eventually  success- 
fully inoculated  into  the  human  conjunctiva.  In  the  same  year 
KartulisJ  in  Alexandria  succeeded  in  cultivating  the  same  organ- 
ism. In  1894  Morax  published  a  brochure  in  Paris  in  which  he 
says  that  "the  disease  [which  he  describes  under  the  name  of  acute 
conjunctivitis]  is  characterized  by  the  constant  presence  in  the 
conjunctival  secretions  of  a  small  bacillus  seen  for  the  first  time  by 
Koch,  but  studied  some  years  later  by  Weeks,  and  now  known  as 
the  bacillus  of  Weeks." 

Further  descriptive  and  clinical  information  can  be  found  in  a 
paper  by  Weeks,  "The  Status  of  our  Knowledge  of  the  ^Etiological 
Factor  in  Acute  Contagious  Conjunctivitis."! 

*  "Wiener  klin.  Wochenschrift,"  1883,  p.  1550. 
t  "N.  Y.  Med.  Record,"  May  21,  1887. 
j  "  Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1887,  p.  289. 

§  "New  York  Eye  and  Ear  Infirmary  Reports,"  Jan.,  1895,  v°l-  m>  Part,  i,  p. 
24. 

406 


Cultivation  407 

Morphology. — The  organism  is  very  tiny  and  is  said  to  bear 
some  resemblance  to  the  bacillus  of  mouse-septicemia.  It  measures 
i  to  2  X  0.25  JJL  (Weeks).  The  length  is  more  constant  in  individuals 
found  in  the  pus  than  those  taken  from  cultures.  In  cultures  the 
organisms  are  longer  and  more  slender.  Involution  forms  of  con- 
siderable length  and  of  irregular  shape  also  occur.  No  spores  are 
observed.  The  organism  has  no  flagella  and  is  not  motile. 

Staining. — Weeks  found  that  the  organism  stained  well  with 
watery  solutions  of  methylene  blue,  basic  fuchsin,  or  gentian 
violet.  The  color  is  fainter  than  that  of  the  nuclei  of  the  associated 
pus-corpuscles,  and  is  much  less  intense  in  old  than  in  fresh  cultures. 
It  is  readily  given  up  when  treated  with  alcohol  or  acids.  Morax 
found  that  the  bacilli  did  not  retain  the  color  in  Gram's  method. 


Fig.  148. — The  Koch- Weeks  bacillus  in  conjunctival  secretion.     Magnified  1000 
diameters  (Rymowitsch  and  Matschinsky). 

Cultivation. — The  organism  refuses  to  grow  upon  any  of  the 
ordinary  culture-media.  Weeks  found,  however,  that  if  the  per- 
centage of  agar-agar  used  was  reduced  to  0.5  per  cent.,  growths 
could  be  secured  by  incubation  at  37°C.,  and  successful  transplanta- 
tions carried  on  to  the  sixteenth  generation.  Abundant  moisture 
was  essential.  The  method  of  isolation  adopted  by  Weeks  was  as 
follows :  « 

"The  conjunctival  sacs  were  thoroughly  washed  with  clean  water,  removing 
the  secretion  present  by  means  of  absorbent  cotton.  The  patient  was  then 
directed  to  keep  the  eyes  closed.  After  five  or  ten  minutes  had  elapsed,  the  eyes 
were  opened,  and  the  secretion  that  had  formed,  lying  at  the  bottom  of  the  cul- 
de-sac,  was  removed  by  means  of  a  sterilized  platinum  rod  and  transferred  to  the 
surface  of  the  agar.  The  mass  of  tenacious  secretion  was  drawn  over  the  surface 
of  the  agar  and  left  there,  the  platinum  being  thrust  into  the  agar  two  or  three 
times  before  removal." 

At  the  end  of  forty-eight  hours  a  slight  haziness  appears  along 
the  path  of  the  wire,  and  on  the  surface  of  the  agar  a  very  small 


408  Acute  Contagious  Conjunctivitis 

patch  is  noticeable;  this  is  of  a  pearly  color  and  possesses  a  glisten- 
ing surface.  By  the  formation  of  small  concentric  colonies  the 
growth  extends  for  a  short  distance.  At  the  end  of  the  fourth  or 
fifth  day  the  growth  ceases  to  advance;  it  is  never  abundant.  The 
culture  dies  in  from  one  to  three  weeks. 

Pathogenesis. — Both  Weeks  and  Morax  have  tested  the  organ- 
ism for  pathogenic  activity,  and  in  every  case  in  which  pure  cultures 
of  it  were  placed  upon  the  human  conjunctiva,  typical  attacks  of 
the  acute  conjunctivitis  resulted.  The  organism  fails  to  infect  any 
of  the  lower  animals. 

Association. — Both  Weeks  and  Morax  found  the  organism  in 
intimate  association  with  a  larger  club-shaped  bacillus,  which 
was  regarded  as  the  pseudo-diphtheria  bacillus.  It  seems  to  be  of 
no  pathogenic  significance. 

THE  MORAX-AXENFELD  BACILLUS 

In  1896  Morax*  found  a  new  bacillus  in  certain  cases  of  epidemic 
subacute  conjunctivitis.  Immediately  afterward  Axenfeld*  pre- 
sented to  a  congress  in  Heidelberg  cultures  of  the  same  bacillus  that 
he  had  isolated  from  51  cases  of  what  he  called  "  Diplobacillen- 
conjunctivitis"  that  occurred  a  few  months  before  as  an  epidemic 
in  Marburg.  De  Schweinitz  and  Veasy,{  Alt§  and  others  found 
the  same  diplobacillus  in  America,  and  many  others  confirmed  the 
observations  in  various  parts  of  Europe.  It  has  also  been  found  in 
Egypt.  There  is  no  doubt,  therefore,  but  that  this  is  a  widely  dis- 
tributed organism.  Morax  produced  the  disease  by  placing  a  pure 
culture  of  the  organism  upon  the  human  conjunctiva.  He  was 
unable  to  infect  any  of  the  lower  animals. 

In  this  subacute  form  of  conjunctivitis  there  is  very  little  secre- 
tion, and  to  secure  the  micro-organism  either  for  microscopic  ex- 
amination or  for  cultivation  recourse  must  be  had  to  minute  flakes 
of  grayish  mucus  that  collect  upon  the  caruncle. 

Morphology. — The  bacillus  is  small,  commonly  occurs  in  pairs 
or  chains.  It  measures  approximately  2  M  in  length.  It  is  not 
motile,  has  no  flagella,  and  forms  no  spores.  It  is  somewhat  pleo- 
morphous.  Involution  forms  soon  appear  in  artificial  cultures. 

Staining. — The  organism  stains  by  ordinary  methods,  but  does 
not  stain  by  Gram's  method. 

Cultivation. — The  organism  grows  only  upon  alkaline  blood- 
serum  or  upon  culture-media  containing  blood-serum.  Morax 
made  his  original  observation  by  using Lb'fBer's  blood-serum  mixture. 
The  colonies  appear  in  twenty-four  hours  at  37°C.  The  blood- 

*"Ann.  de  1'Inst.  Pasteur,"  June,  1896;  "Ann.  d'Oculist,"  Jan.,  1897. 
f"  Heidelberg  Congress,"  1 896;  "Centralbl.  f.  Bakt,"  etc.,  1897,  xxi. 
j  "Ophthalmological  Record,"  1899. 
§  "  Amer.  Jour,  of  Ophthalmology,"  1898,  p.  171. 


Zur  Nedden's  Bacillus  409 

serum  is  almost  immediately  liquefied,  so  that  the  growing  colonies 
appear  to  be  sinking  into  the  medium  after  thirty-six  hours.  The 
entire  tube  of  medium  may  eventually  be  liquefied. 

Upon  agar-agar  containing  serum,  grayish-white  colonies  of 
small  size,  resembling  colonies  of  gonococci,  are  formed.  Growth 
is  slow.  Bouillon  is  slowly  clouded. 

Pathogenesis. — The  pathogenic  and  specific  nature  of  the  diplo- 
bacillus  was  made  clear  by  Morax,  who  produced  the  disease  in 
man  by  placing  a  pure  culture  upon  the  human  conjunctiva. 

ZUR  NEDDEN'S  BACILLUS 

This  bacillus  was  the  only  organism  that  Haupt*  was  able  to 
isolate  from  a  neuroparalytic  with  confluent  peripheral  ulcera- 


, 


Fig.   149. — The    Morax-Axenfeld    diplobacillus    of    conjunctivitis.     Magnified 
1000  diameters  (Rymowitsch  and  Matschinsky). 

tions  of  the  cornea.  It  seemed  to  be  identical  with  an  organism 
that  zur  Nedden  had  found  previously  in  a  case  of  corneal  ulcera- 
tion  in  the  clinic  at  Bonn. 

Morphology. — It  is  a  tiny  bacillus,  less  than  i  M  in  length,  slightly 
curved,  generally  single,  but  sometimes  in  pairs  and  short  chains. 
It  is  not  motile,  has  no  flagella,  forms  no  spores. 

Staining. — It  stains  ordinarily,  but  not  by  Gram's  method. 

Cultivation. — It  is  easily  cultivated  upon  the  ordinary  laboratory 
media,  the  cultures  being  without  characteristic  peculiarities. 
Gelatin  is  not  liquefied.  Milk  is  coagulated.  Acid  but  no  gas  is 
formed  in  glucose  media.  A  thick  yellowish  growth  appears  upon 
potato.  No  indol  is  formed. 

Pathogenesis. — Corneal  ulcers  were  formed  in  a  guinea-pig 
after  artificial  implantation  in  the  corneal  tissue. 

*"  Inaugural  Dissertation,"  Bonn,  1902. 


4i o  Acute  Contagious  Conjunctivitis 


MISCELLANEOUS  ORGANISMS  IN  CONJUNCTIVITIS 

In  addition  to  the  foregoing  organisms,  others  not  infrequently 
make  their  appearance  as  excitants  of  conjunctivitis.  The  most 
frequent  of  these  being  the  pneumococcus,  the  most  dangerous,  the 
gonococcus.  The  former  produce  a  severe  conjunctivitis,  with 
the  formation  of  a  false  membrane,  the  latter  the  well-known 
blenorrhea  and  ophthalmia  neonatorum.  Streptococci,  diphtheria 
bacilli,  staphylococci,  meningococci,  colon  bacilli,  Bacillus  pneumonia 
(Friedlander) ,  and  other  organisms  have  been  found  and  appear 
to  be  responsible  for  conjunctivitis 


CHAPTER  XII 
DIPHTHERIA 

BACILLUS  DIPHTHERIA  (KLEBS-LOFFLER) 

General  Characteristics. — A  non-motile,  non-flagellate,  non-sporogenous, 
non-chromogenic,  non-liquefying,  aerobic,  purely  parasitic,  pathogenic,  toxico- 
genic  bacillus,  cultivable  upon  the  ordinary  culture  media,  staining  by  the  ordi- 
nary methods  and  by  Gram's  method. 

In  1883  Klebs*  demonstrated  the  presence  of  a  bacillus  in  the 
pseudo-membranes  upon  the  fauces  of  patients  suffering  from 
diphtheria,  but  it  was  not  until  1884  tnat  LofHerf  succeeded  in 
isolating  and  cultivating  it.  The  organism  is  now  known  by  both 
their  names,  and  called  the  Klebs-Loffler  bacillus. 

Morphology. — The  bacillus  is  about  the  length  of  the  tubercle 
bacillus  (1.5-6.5  AI),  but  about  twice  its  diameter  (0.4-1.0  M),  has  a 
slight  curve  similar  to  that  which  characterizes  the  tubercle  bacillus, 
and  has  rounded  and  usually  clubbed  ends.  It  does  not  form 
chains,  though  two,  three,  and  rarely  four  individuals  may  be  found 
conjoined;  usually  the  individuals  are  separate  from  one  another. 
The  bacillus  has  no  flagella,  it  is  non-motile,  and  does  not  form  spores. 
Distinct  polar  granules  can  be  defined  at  the  ends  of  the  bacilli. 
Occasional  branched  forms  are  observed,  though  Abbott  and  Gilder- 
sleeve:!:  do  not  regard  branching  as  a  phase  of  the  normal  develop- 
ment of  the  organism  and  do  not  find  it  common  upon  the  standard 
culture  media.  The  bacillus  is  peculiar  in  its  pleomorphism,  for 
among  the  well-formed  individuals  which  abound  in  fresh  cultures  a 
large  number  of  peculiar  organisms  are  to  be  found,  much  larger 
than  normal,  some  with  one  end  enlarged  and  club  shaped,  some 
greatly  elongated,  with  both  ends  similarly  and  irregularly  expanded. 
These  probably  represent  an  involution  form  of  the  organism,  for 
they  are  present  in  perfectly  fresh  cultures. 

The  involution  of  the  diphtheria  bacillus  seems  to  occur  in  pro- 
portion to  the  rapidity  of  its  growth.  Upon  Loffler's  serum  mix- 
ture, which  seems  best  adapted  for  its  cultivation,  the  involution 
of  the  organism  takes  place  with  great  rapidity,  so  that  large  clubbed 
organisms  and  large  organisms  with  polar  granules  are  very  common. 
On  the  other  hand,  upon  agar  and  glycerin  agar-agar,  where  the 
organism  grows  very  slowly,  it  usually  appears  in  the  form  of 
short  spindle  and  lancet  shapes.  So  different  are  these  forms  that 

"Verhandlungen  des  Congresses  fur  innere  Med.,"  1883. 
t  "Mittheilungen  aus  dem  kaiserlichen  Gesundheitsamte,"  2. 
j  "Centralbl.  f.  Bakt,"  etc.,  Dec.  18,  1903,  Bd.  xxxv,  No.  3. 
411 


412 


Diphtheria 


"'' 


Fig.  150. — Bacillus  diphtheriae,  five  hours  Fig.  151. — Bacillus  diphtherias,  same  cul- 

at  36°  C.    This   shows  only   solid   staining        ture,  eight  hours  at  36°  C.     This  also  shows 
forms.  solid  forms,  many  of  them  with  parallel  ar- 

rangement. 


Fig.  1 5  2. — Bacillus  diphtheriae,  same  cul- 
ture, twelve  hours  at  36°  C.  The  bacilli 
stain  faintly  at  their  ends,  and  in  some  small 
granules  are  seen  at  the  tip  of  the  faintly 
stained  portions. 


Fig.  153- — Bacillus  diphtheriae,  same  cul- 
ture, fifteen  hours  at  36°  C.  The  bacilli 
stain  more  unevenly  and  the  granules  are 
larger. 


.'•<?•-.. 

./•?•'  -'V 


Fig.  154- — Bacillus  diphtheriae,  same  cul-  Fig.    155.— Bacillus    diphtheriae,    forty- 

ture,    twenty-four    hours   at   36°   C.    This  eight   hours   at   36°  C.     This   is   the   same 

shows  clubbed  and  barred  forms  as  well  as  bacillus  as  in  the  preceding  figures,  but  from 

granular  forms.     At  the  lower  part  of  the  a  culture  where  the  colonies  were  discrete, 

field  is  a  paired  form  which  shows  the  char-  It  shows  long  filamentous  forms, 
acteristic  clubbing  of  the  distal  ends. 

(Photomicrographs  by  Mr.  Louis  Brown.  The  magnification  is  the  same  in  all — X  2000. 
All  of  the  preparations  were  made  from  growth  on  blood-serum.)  (Francis  P.  Denny,  in 
"  Jour,  of  Med.  Research."; 


Cultivation  413 

a  beginner  would  certainly  fail  to  recognize  them  as  the  same  species. 
The  small  short  forms  also  stain  much  more  uniformly  than  the 
large  club-shaped  bacilli. 

Staining. — The  bacillus  can  readily  be  stained  with  aqueous 
solutions  of  the  anilin  colors,  but  more  characteristically  with 
Loffler's  alkaline  methylene  blue: 

Saturated  alcoholic  solution  of  methylene  blue 30 

i:  10,000  aqueous  solution  of  caustic  potash 100 

Emery  prefers  Manson's  borax  methylene  blue.  A  stock  solu- 
tion which  keeps  well  is  prepared  by  dissolving  2  grams  of  meth- 
ylene blue  and  5  grams  of  borax  in  100  cc.  of  water.  This  is  diluted 
with  from  five  to  ten  times  its  volume  of  water  for  ordinary  use. 
An  aqueous  solution  of  dahlia  is  recommended  by  Roux. 

The  Neisser  method  of  staining  the  diphtheria  bacillus,  which  met 
with  a  very  cordial  reception,  is  as  follows: 

The  prepared  cover-glass  is  immersed  for  from  two  to  three 
seconds  in 

Alcohol  (96  per  cent.) 20  parts 

Methylene  blue i  part 

Distilled  water 950  parts 

Acetic  acid  (glacial) 50  parts 

Then  for  three  to  five  seconds  in 

Bismarck  brown i  part 

Boiling  distilled  water £00  parts 

The  true  diphtheria  bacilli  appear  brown,  with  a  dark  blue  body 
at  one  or  both  ends;  the  pseudo-diphtheria  bacilli  usually  exhibit 
no  polar  bodies. 

Park*  found  that  neither  the  Neisser  nor  the  Roux  stain  gave  any 
more  information  concerning  the  virulence  of  the  bacilli  than  the 
Loffler  alkaline  methylene  blue. 

The  bacilli  stain  well  by  Gram's  method,  which  is  excellent  for 
their  definition  in  sections  of  tissue,  though  Welch  and  Abbott  found 
that  Weigert's  fibrin  method  and  picrocarmin  gave  the  most  beau- 
tiful results. 

Cultivation. — The  diphtheria  bacillus  grows  readily  upon  all 
the  ordinary  media,  and  is  very  easy  to  obtain  in  pure  culture, 
plates  not  being  necessary.  It  is  purely  aerobic. 

Colonies. — Upon  the  surface  of  gelatin  plates  the  colonies  attain 
but  a  small  size  and  appear  to  the  naked  eye  as  whitish  points  with 
smooth  contents  and  regular,  though  sometimes  indented,  borders. 
Under  the  microscope  they  appear  granular  and  yellowish-brown, 
with  irregular  borders.  Upon  agar-agar  and  glycerin  agar-agar  the 
colonies  are  slower  to  develop,  larger,  more  translucent,  without  the 
yellowish-white  or  china-white  color  of  the  blood-serum  cultures, 
and  are  more  or  less  distinctly  divided  into  a  small  elevated  center 
*  "Bacteriology  in  Medicine  and  Surgery,"  1900. 


414  Diphtheria 

and  a  flat  surrounding  zone  with  indented  edges,  and  a  radiated 
appearance.  The  colonies  that  develop  upon  Loffler's  blood-serum 
mixture  are  rounded,  yellowish-white,  good  sized  and  more  or  less 
confluent  when  closely  approximated.  They  are  smooth,  moist  and 
shining  on  the  surface.  They  are  with  difficulty  differentiated  from 
those  of  Bacillus  hofmanni,  the  pseudo-diphtheria  bacillus. 

Gelatin. — The  growth  in  gelatin  puncture  is  scanty,  not  char- 
acteristic, and  consists  of  small  spheric  colonies  along  the  line  of 
inoculation.  The  gelatin  is  not  liquefied. 


Fig.  156. — Diphtheria  bacilli  (from  photographs  taken  by  Prof.  E.  K.  Dun- 
ham, Carnegie  Laboratory,  New  York):  a,  Pseudobacillus;  b,  true  bacillus;  c, 
pseudobacillus. 

Agar-agar. — Cultures  upon  the  surface  of  agar-agar  slants  are 
usually  meager  when  contracted  with  those  upon  LofBer's  blood- 
serum  mixture,  and  may  be  whitish  in  color.  They  consist  of  dis- 
crete and  confluent  whitish  colonies  devoid  of  differential  qualities. 
The  oftener  the  organism  is  transplanted  to  fresh  agar-agar,  the 
more  luxuriant  its  growth  becomes.  The  growth  is  rapid  and  lux- 
uriant upon  glycerin  agar-agar. 

Bouillon. — When  planted  in  bouillon  a  distinct,  whitish,  granular 
pellicle  forms  upon  the  surface  of  the  clear  medium.  The  pellicle 
appears  quite  uniform  when  the  tube  or  flask  is  undisturbed,  but  it 
is  so  brittle  that  it  at  once  falls  to  pieces  if  disturbed,  the  minute 


Cultivation  415 

fragments  slowly  sedimenting  and  forming  a  miniature  snow-storm 
in  the  flask  or  tube.  The  organism  at  times  also  causes  a  diffuse 
cloudiness  of  the  medium,  but,  not  being  motile,  soon  settles  to  the 
bottom  in  the  form  of  a  flocculent  precipitate  which  has  a  tendency 
to  cling  to  the  sides  of  the  glass,  and  leave  the  bouillon  clear. 

No  fermentation  occurs  in  bouillon  to  which  sugar  is  added,  though 
acids  are  soon  formed  by  which  the  growth  is  checked.  If,  how- 
ever, the  quantity  of  sugar  be  too  small  to  check  the  growth,  the 
acidity  gives  place  to  increasing  alkalinity  at  a  later  period. 


Fig.  157. — Bacillus  diphtherias;  colony  twenty-four  hours  old,  upon  agar-agar 
Xioo  (Frankel  and  Pfeiffer). 

Spronck*  found  that  the  characteristics  of  the^  growth  of  the 
diphtheria  bacilljas  in  bouillon,  as  well  as  the  amount  of  toxin 
produced,  vary  according  to  the  amount  of  glucose  in  the  bouillon. 

Zinnof  found  that  digested  brain  added  to  the  culture  bouillon 
greatly  facilitated  the  growth  of  diphtheria  and  tetanus  bacilli  and 
increased  the  toxin-production. 

Blood-serum. — The  bacillus  grows  similarly  upon  blood-serum 
and  LorBer's  mixture,  but  more  luxuriously  upon  the  latter,  where 
large,  creamy- white,  discrete  and  confluent,  moist,  shining  colonies 
form.  The  rapidity  of  the  growth  which  is  abundant  in  twenty- 
four  hours,  and  the  appearances  presented  are  quite  characteristic. 

Loffler  has  shown  that  the  addition  of  a  small  amount  of  glucose 
to  the  culture-medium  increases  the  rapidity  of  growth,  and  suggests 
a  special  medium  which  bears  his  name — Loffler's  blood-serum 
mixture: 

Blood-serum 3 

Ordinary  bouillon  +  i  per  cent,  of  glucose i 

*"Ann.  de  1'Inst.  Pasteur,"  Oct.  25,  1895,  vol.  ix,  No.  10,  p.  758. 
t  "Centralbl.  f.  Bakt.,"  Jan.  4,  1902,  xxxi,  No.  2,  p.  42. 


4i  6  Diphtheria 

This  mixture  is  filled  into  tubes,  coagulated,  and  sterilized  like 
blood-serum,  and  is  one  of  the  best  known  media  to  be  used  in  con- 
nection with  the  study  of  diphtheria. 

Material  from  the  infected  throat  can  be  taken  with  a  swab  or 
platinum  loop  and  spread  upon  the  surface  of  several  successive 
tubes  of  Loffler's  blood-serum  media.  Upon  the  first  a  confluent 
growth  of  the  bacillus  usually  occurs;  but  upon  the  third,  scattered 
cream-white  colonies  suitable  for  transplantation  can  usually  be 
found. 

The  studies  of  Michel*  have  shown  that  the  development  of 
the  culture  is  much  more  luxuriant  and  rapid  when  horses'  serum 
instead  of  beef  or  calves'  serum  is  used. 

Westbrook  suggested  that  the  addition  of  a  small  amount  of 
glycerin  to  the  preparation  of  blood-serum  would  prevent  it  from 
drying  so  rapidly  as  usual  and  would  have  the  added  advantage  of 
preventing  the  growth  of  certain  varieties  of  bacteria  not  desired. 
Dubois  I  carried  out  a  series  of  observations  upon  this  question 
and  found  that  3  to  5  per  cent,  of  glycerin  makes  a  very  valu- 
able addition,  as  the  diphtheria  bacilli  grow  very  rapidly  and 
almost  in  pure  culture  upon  the  blood-serum  mixture  to  which 
it  is  added.  The  blood  serum  is  not  liquefied  or  otherwise  visibly 
changed. 

Potato. — Upon  potato  it  develops  only  when  the  reaction  is 
alkaline.  The  potato  growth  is  not  characteristic. 

Milk. — Milk  is  an  excellent  medium  for  the  cultivation  of  Bacillus 
diphtherias.  The  milk  is  not  coagulated.  Litmus  milk  is  useful 
for  detecting  the  changes  of  reaction  brought  about.  Alkalinity, 
which  at  first  favors  the  development  of  the  bacillus,  is  soon  replaced 
by  acidity  that  checks  it.  When  the  culture  becomes  old,  the  reac- 
tion may  again  become  strongly  alkaline.  This  variation  in  reaction 
seems  to  depend  entirely  on  the  transformation  of  sugar  in  the 
media. 

Vital  Resistance. — As  the  diphtheria  bacillus  does  not  form  spores, 
it  possesses  very  little  vital  resistance  and  is  delicate  in  its  thermic 
sensitivity.  It  grows  slowly  at  2o°C.,  rapidly  at  37°C.,  and  ceases 
to  grow  at  about  4o°C.  It  is  killed  when  exposed  to  58°C.  for  a 
few  minutes.  Besson  states  that  when  dried  in  fragments  of  false 
membrane  it  resists  high  temperatures  and  has  been  found  alive 
after  exposure  to  ioo°C.  for  an  hour.  Drying  quickly  destroys  it, 
but  if  organic  matter  be  present  it  may  remain  alive  a  long  time. 
Roux  and  Yersin  were  able  to  keep  the  bacilli  alive  in  a  piece  of  dry 
pseudo-membrane,  kept  in  the  dark,  for  five  months. 

Reyes  has  demonstrated  that  in  absolutely  dry  air  diphtheria 
bacilli  die  in  a  few  hours.  Under  ordinary  conditions  their  vitality, 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Sept.  24,  1897,  Bd.  xxn,  Nos.  10  and  n. 
f  "Seventeenth  Annual  Report  of  the  Department  of  Health  and  Charities," 
Indianapolis,  Ind.,  1907. 


Metabolic  Products  417 

when  dried  on  paper,  silk,  etc.,  continues  for  but  a  few  days,  though 
sometimes  they  can  live  for  several  weeks.  In  sand  exposed  to  a 
dry  atmosphere  the  bacilli  die  in  five  days  in  the  light;  in  sixteen 
to  eighteen  days  in  the  dark.  When  the  sand  is  exposed  to  a  moist 
atmosphere,  the  duration  of  their  vitality  is  doubled.  In  fine 
earth  they  remained  alive  seventy-five  to  one  hundred  and  five 
days  in  dry  air,  and  one  hundred  and  twenty  days  in  moist  air. 

The  organism  is  highly  susceptible  to  disinfectantsexc  ept  when 
dried  in  false  membrane. 

Metabolic  Products. — The  diphtheria  bacillus  forms  acids  (lactic 
acid?)  in  the  presence  of  dextrose,  galactose,  levulose,  maltose, 
dextrine  and  glycerin.  It  also  forms  acids  in  meat-infusion  bouillon, 
probably  because  of  the  muscle  sugars  it  contains.  In  the  absence  of 
sugars  it  produces  alkalies.  It  is  unable  to  evolve  gas  from  any 
carbohydrates.  It  does  not  coagulate  milk;  does  not  liquefy 
gelatin  or  blood-serum. 

Palmirski  and  Orlowski*  assert  that  the  bacillus  produces  indol, 
but  only  after  the  third  week.  Smith,!  however,  found  that  when 
the  diphtheria  bacillus  grew  in  dextrose-free  bouillon  no  indol  was 
produced. 

Toxin. — The  earliest  researches  upon  the  nature  of  the  poisonous 
products  of  the  diphtheria  bacillus  seem  to  have  been  made  in  1887 
by  Loffler,t  who  came  to  the  conclusion  that  they  belonged  to  the 
enzymes.  The  credit  of  removing  the  bacteria  from  the  culture  by 
filtration  through  porcelain  and  the  demonstration  of  the  soluble 
poison  in  the  filtrate  belong  to  Roux  and  Yersin.§  Toxic  bouillon 
prepared  in  this  manner  was  found  to  cause  serous  effusions  into 
the  pleural  cavities,  acute  inflammation  of  the  kidneys,  fatty  de- 
generation of  the  liver,  and  edema  of  the  tissue  into  which  the 
injection  was  made.  In  some  cases  palsy  subsequently  made  its 
appearance,  usually  in  the  hind  quarters.  The  effect  of  the  poison 
was  slow  and  death  took  place  days  or  weeks  after  injection, 
sometimes  being  preceded  by  marked  emaciation.  Temperatures 
of  58°C.  lessened  the  activity  of  the  toxin  and  temperatures  of 
ioo°C.  destroyed  it.  It  was  precipitated  by  absolute  alcohol  and 
mechanically  carried  down  by  calcium  chlorid.  Brieger  and 
Frankel||  confirmed  the  work  of  Roux  and  Yersin,  and  concluded 
that  the  poison  was  a  toxalbumin.  Tangl**  was  able  to  extract  the 
toxin  from  a  fragment  of  diphtheria  pseudo-membrane  macerated 
in  water. 

The  nature  of  the  diphtheria  toxin  has  been  studied  by  Ehrlichf  t 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  March,  1895. 

t  "Jour.  Exp.  Med.,"  Sept.,  1897,  vol.  n,  No.  5,  p.  546. 

t  "Centralbl.  f.  Bakt.,"  etc.,  1887,  n,  p.  105. 

§  "Ann.  de  1'Inst.  Pasteur,"  1888-1889. 

||  "Berliner  klin.  Wochenschrift,"  1890,  11-12. 
**  "Centralbl.  f.  Bakt.,"  etc.,  Bd.  xi,  p.  379. 
tt"Klinisches  Jahrbuch,"  1897. 
27 


4i  8  Diphtheria 

and  found  to  be  extremely  complex.  As  it  exists  in  cultures  it  is 
composed  of  equal  parts  of  toxin  and  toxoid.  Of  these,  the  former 
is  poisonous,  the  latter  harmless  for  animals — or  at  least  not  fatal 
to  them.  The  toxoids  have  equal  or  greater  affinity  for  combining 
with  antitoxin  than  the  toxin  and  cause  confusion  in  testing  the 
unit  value  or  strength  of  the  antitoxin.  In  old  or  heated  toxin  all 
of  the  toxin  molecules  become  changed  into  toxons  or  toxoids  and 
the  poisonous  quality  is  lost  though  the  power  of  combining  with 
antitoxin  remains. 

The  toxin  is  extremely  poisonous,  and  a  filtered  bouillon  con- 
taining it  may  be  fatal  to  a  3oo-gram  guinea-pig  in  doses  of  only 
0.0005  cc.  It  is  thought  not  to  be  an  albuminous  substance,  as 
it  can  be  elaborated  by  the  bacilli  when  grown  in  non-albuminous 
urine,  or,  as  suggested  by  Uschinsky,  in  non-albuminous  solutions 
whose  principal  ingredient  is  asparagin.  The  toxic  value  of  the 
cultures  is  greatest  in  the  second  week. 

This  soluble  toxin  so  well  known  in  bouillon  cultures  is  probably 
only  one  of  the  poisonous  substances  produced  by  the  bacillus.  An 
intracellular,  insoluble  toxic  product  seems  to  have  been  discovered 
by  Rist,*  who  found  it  in  the  bodies  of  dried  bacilli,  and  observed 
that  it  was  not  neutralized  by  the  antitoxin. 

Pathogenesis. — The  Bacillus  diphtherias  is  pathogenic  for  man, 
monkeys,  guinea-pigs,  rabbits,  dogs,  cats,  cows,  and  horses.  Spar- 
rows, pigeons  and  fowls  are  susceptible  to  experimental  infection; 
rats  and  mice  are  immune.  Spontaneous  or  natural  infection  is 
pretty  well  limited  to  man.  The  effects  of  artificial  experimental 
infection  vary  with  the  avenue  of  infection,  the  quantity  of  culture 
and  its  virulence. 

1.  Subcutaneous  inoculation  in  rabbits  and  guinea-pigs  is  usually 
fatal  in  from  seventy-two  hours  to  five  days.     The  animal  suffers 
some  rise  of  temperature  in   twelve  to  twenty-four  hours,  soon  is 
depressed,  weak,  loses  flesh,  remains  quiet  and  dies.     At  the  seat 
of  infection  there  is  a  swelling  caused  by  combined  edema,  hemor- 
rhage and  fibrinous  exudation.     If  the  culture  be  of  feeble  viru- 
lence so  that  death  does  not  occur,  this  area  sloughs,  and  then 
heals  slowly. 

2.  Intraperitoneal  and  Intrapleural  Infection. — This  is  not  so 
serious  in  its  results  as  might  be  supposed.     Some  animals  recover 
from  doses  that  might  be  fatal  under  the  skin.     Death  does  not 
occur  until  after  a  week  or  twelve  days.     Fluid  of  slightly  turbid 
character  with  flakes  of  fibrin  is  found  in  the  peritoneum. 

3.  Mucous  Membrane  Inoculations. — When  implanted  upon  the 
scarified  surfaces  of  the  mucous  membranes,  the  bacillus  causes  the 
formation  of  a  fibrinous  and  necrotic  pseudo-membrane.     Such  con- 
ditions may  recover  or  death  may  follow  after  some  days. 

In  all  cases  the  bacilli  remain  fairly  well-localized  at  or  near  the 
*  "Soc.  de  Biol.  Paris,"  1903,  No.  25. 


Pathogenesis  419 

seat  of  inoculation  and  only  rarely  invade  the  blood.  Death  and 
illness  result  from  toxemia,  not  from  bacteremia. 

When  examined  post-mortem,  the  liver  is  found  enlarged  and 
sometimes  shows  minute  whitish  points,  which  upon  microscopic 
examination  prove  to  be  necrotic  areas  in  which  the  cells  are  com- 
pletely degenerated,  and  the  chromatin  of  their  nuclei  scattered 
about  in  granular  form.  Similar  necrotic  foci,  to  which  attention 
was  first  called  by  Oertel,  are  present  in  nearly  all  the  organs  in 
cases  of  death  from  diphtheria  intoxication.  No  bacilli  are  present 
in  these  lesions.  Welch  and  Flexner*  have  shown  these  foci  to 
be  common  to  numerous  intoxications,  and  riot  peculiar  to  diphtheria. 

The  lymphatic  glands  are  usually  enlarged,  and  the  adrenals 
enlarged  and  hemorrhagic.  The  kidneys  show  parenchymatous 
degeneration. 

Roux  and  Yersin  found  that  when  the  bacilli  were  introduced 
into  the  trachea  of  animals,  a  typical  pseudo-membrane  was 
formed,  and  that  diphtheritic  palsy  sometimes  followed. 

Diphtheria  in  man  is  characterized  by  a  pseudo-membranous  in- 
flammation of  the  mucous  membranes,  particularly  of  the  fauces, 
though  it  may  occur  in  the  nose,  in  the  mouth,  upon  the  genital 
organs,  or  upon  wounds.  Williams!  has  reported  a  case  of  diph- 
theria of  the  vulva,  and  Nisot  and  Bumm  have  reported  cases  of 
puerperal  diphtheria  from  which  the  bacilli  were  cultivated.  It  is 
in  nearly  all  cases  a  purely  local  infection,  depending  upon  the  pres- 
ence and  development  of  the  bacilli  upon  the  diseased  mucous  mem- 
brane, but  is  accompanied  by  a  serious  intoxication  resulting  from 
the  absorption  from  the  local  lesions  of  a  poisonous  metabolic  product 
of  the  bacilli.  The  bacilli  are  found  only  in  the  membranous  exuda- 
tion, and  are  most  plentiful  in  its  older  portions. 

The  entrance  of  the  diphtheria  bacillus  into  the  internal  organs 
can  scarcely  be  regarded  as  a  frequent  occurrence,  though  metastatic 
occurrence  of  the  organism  with  and  without  associated  staphylococci 
and  streptococci,  and  with  and  without  purulent  inflammations  have 
from  time  to  time  been  reported.  Diphtheria  bacilli  were  first 
found  in  the  heart's  blood,  liver,  spleen,  and  kidney,  by  Frosch.J 
Kolisko  and  Paltauf||  had  already  found  them  in  the  spleen,  and 
other  observers  in  various  lesions  of  the  deeper  tissues  and  oc- 
casionally in  the  organs.  In  the  blood  and  organs  it  is  commonly 
associated  with  Streptococcus  pyogenes  and  sometimes  with  other 
bacteria.  While  present  in  nearly  all  of  the  inflammatory  sequelae 
of  diphtheria,  the  Klebs-Loffler  bacillus  probably  has  very  little  in- 
fluence in  producing  them,  the  conditions  being  almost  invariably 
associated  with  the  pyogenic  cocci,  either  the  streptococci  or 

'  "Bull,  of  the  Johns  Hopkins  Hospital,"  Aug.,  1901. 

t  "Amer.  Jour,  of  Obstet.  and  Dis.  of  Women  and  Children,"  Aug.,  1898. 
j  "Zeitschrift  fur  Hygiene,"  etc.,  1893,  xm,  Heft  i. 
I!  "Wiener  klin.  Wochenschrift,"  1880. 


42O  Diphtheria 

staphylococci.  Howard*  studied  a  case  of  ulcerative  endocarditis 
caused  by  the  diphtheria  bacillus,  and  Pearcef  has  observed  it  in 
i  case  of  malignant  endocarditis,  19  out  of  24  cases  of  broncho- 
pneumonia,  i  case  of  empyema,  16  cases  of  middle-ear  disease,  8 
cases  of  inflammation  of  the  antrum  of  Highmore,  i  case  of  in- 
flammation of  the  sphenoidal  sinuses,  i  case  of  thrombosis  of  the 
lateral  sinuses,  2  cases  of  abscesses  of  the  cervical  glands,  and  in 
esophagitis,  gastritis,  vulvo-vaginitis,  dermatitis,  and  conjunctivitis 
following  or  associated  with  diphtheria. 

A  case  of  septic  invasion  by  the  diphtheria  bacillus  is  reported 
by  Ucke,{  who  gives  a  synopsis  of  the  literature  of  similar  cases. 

The  disease  pursues  a  variable  course.  In  favorable  cases  the 
patient  recovers  gradually,  the  pseudo-membrane  first  disappearing, 
leaving  an  inflamed  mucous  membrane,  upon  which  virulent  diph- 
theria bacilli  persist  for  weeks  and  sometimes  for  months.  Smith* 
describes  the  bacteriologic  condition  of  the  throat  in  diphtheria 
as  follows:  "The  microscope  informs  us  that  during  the  earli- 
est local  manifestations  the  usual  scant  miscellaneous  bacterial 
flora  of  the  mucosa  is  quite  suddenly  replaced  by  a  rich  vege- 
tation of  the  easily  distinguishable  diphtheria  bacillus.  Frequently 
no  other  bacteria  are  found  in  the  culture-tube.  This  vegeta- 
tion continues  for  a  few  days,  then  gradually  gives  way  to 
another  flora  of  cocci  and  bacilli,  and  finally  the  normal  condition 
is  reestablished." 

Associated  Bacteria. — Streptococcus  pyogenes  and  Staphylococci 
pyogenes  aureus  and  albus  are,  in  many  cases,  found  in  associa- 
tion with  the  diphtheria  bacillus,  especially  when  severe  lesions  of 
the  throat  exist. 

In  a  series  of  234  cases  carefully  and  statistically  studied  by 
Blasi  and  Russo-Travali,||  it  was  found  that  in  26  cases  of  pseudo- 
membranous  angina  due  to  streptococci,  staphylococci,  colon  bacilli, 
and  pneumococci,  2  patients  died,  the  mortality  being  3.84  per 
cent.  In  102  cases  of  pure  diphtheria,  28  died,  a  mortality  of  27.45 
per  cent.  Seventy-six  cases  showed  diphtheria  bacilli  and  staph- 
ylococci; of  these,  25,  or  32.89  per  cent.,  died.  Twenty  cases 
showed  the  diphtheria  bacilli  and  Streptococcus  pyogenes,  with  6 
deaths — 30  per  cent.  In  7  cases,  of  which  3,  or  43  per  cent.,  were 
fatal,  the  diphtheria  bacillus  was  in  combination  with  streptococci 
and  pneumococci.  The  most  dangerous  forms  met  were  3  cases, 
all  fatal,  in  which  the  diphtheria  bacillus  was  found  in  combination 
with  Bacillus  coli. 

In  157  cases  of  diphtheria  and  scarlatina  studied  at  the  Boston 

*  "Amer.  Jour.  Med.  Sci.,"  Dec.,  1894. 
t  "Jour.  Boston  Soc.  of  Med.  Sci.,"  March,  1898. 

I  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  original,  XLVI,  Heft  4,  March  10,  1908, 
p.  292. 

"Ann.  de  PInst.  Pasteur,"  1896,  p.  387. 


Pathogenesis  421 

City  Hospital  by  Pearce,*  there  were  94  cases  of  diphtheria,  46 
cases  of  complicated  diphtheria  (29  with  scarlet  fever,  n  with  measles, 
and  5  with  measles  and  scarlet  fever),  and  17  cases  of  scarlet  fever 
(in  3  of  which  measles  was  also  present). 

Of  the  94  cases  of  uncomplicated  diphtheria,  the  Klebs-Loffler 
bacilli  were  present  in  the  heart's  blood  in  4,  twice  alone  and  twice 
with  streptococci.  In  9  cases  the  streptococcus  occurred  alone;  in 
i  case  the  pneumococcus  occurred  alone.  In  the  liver  the  bacillus 
was  found  in  24  cases,  alone  in  12  and  together  with  the  strepto- 
coccus in  12;  the  streptococcus  occurred  in  27  cases,  alone  in  14, 
with  the  Klebs-Loffler  bacillus  in  12,  and  with  Staphylococcus 
pyogenes  aureus  in  i.  Staphylococcus  pyogenes  aureus  occurred 
in  4  cases,  alone  in  3  and  associated  with  the  streptococcus  in  i. 
The  pneumococcus  occurred  alone  in  i  case. 

In  the  spleen  the  Klebs-Loffler  bacillus  occurred  eighteen  times, 
fifteen  times  alone  and  three  times  associated  with  the  streptococcus. 
The  streptococcus  occurred  in  24  cases,  alone  in  21,  associated  with 
the  Klebs-Loffler  bacillus  twice,  and  with  Staphylococcus  pyogenes 
aureus  once.  Staphylococcus  pyogenes  occurred  twice,  once  alone 
and  once  with  the  streptococcus.  The  pneumococcus  occurred 
twice  alone. 

In  the  kidney  the  Klebs-Loffler  bacillus  occurred  in  23  cases, 
in  15  alone,  in  5  associated  with  the  streptococcus,  and  in  2  with 
Staphylococcus  pyogenes  aureus.  The  streptococcus  occurred  in 
26  cases,  in  19  of  which  it  was  the  only  organism  present.  Staphyl- 
ococcus pyogenes  aureus  occurred  in  8  cases,  in  4  of  which  it  was 
in  pure  culture.  The  pneumococcus  occurred  four  times,  three  times 
in  pure  culture  and  once  with  the  Klebs-Loffler  bacillus. 

In  the  46  cases  of  complicated  diphtheria,  the  heart's  blood  showed 
pure  cultures  of  the  streptococcus  nine  times  and  the  streptococcus 
associated  with  the  Klebs-Loffler  bacillus  once.  The  diphtheria 
bacillus  occurred  alone  once. 

In  the  liver,  in  10  cases  streptococcus  occurred  alone,  in  7  cases 
associated  with  the  Klebs-Loffler  bacillus,  and  in  3  cases  with 
Staphylococcus  pyogenes  aureus.  The  diphtheria  bacillus  occurred 
in  pure  culture  in  5  cases. 

The  spleen  contained  streptococci  only  thirteen  times  and  mixed 
with  the  diphtheria  bacillus  twice.  The  diphtheria  bacillus  was 
found  in  pure  culture  in  5  cases. 

The  kidney  contained  pure  cultures  of  streptococci  in  10  cases, 
streptococci  associated  with  diphtheria  bacilli  five  times,  and  with 
Staphylococcus  pyogenes  aureus  three  times.  The  diphtheria 
bacillus  occurred  alone  in  7  cases.  Staphylococcus  pyogenes 
aureus  and  the  pneumococcus  each  alone  once,  and  both  together 
once. 

"The  clinical  significance  of  this  general  infection  with  the  Klebs- 
*  "Jour.  Boston  Soc.  of  Med.  Sci.,"  March,  1898. 


422  Diphtheria 

Loffler  bacillus  is  not  apparent.  It  occurred  generally,  but  not 
always,  in  the  gravest  cases,  or  those  known  as  'septic'  cases.  It 
is  probable  that  it  may  be  due  to  a  diminished  resistance  to  the 
tissue-cells,  or  of  the  germicidal  power  of  the  blood.  In  this  series 
of  fatal  cases  the  number  of  infections  with  the  streptococcus  and 
with  the  Klebs-Loffler  bacillus  was  about  even,  though  slightly  in 
favor  of  the  streptococcus." 

The  mixed  infections  add  to  the  clinical  diphtheria  the  patho- 
genic effects  of  the  associated  bacteria.  The  diphtheria  bacillus 
probably  begins  the  process,  growing  upon  the  mucous  membrane, 
devitalizing  it  by  its  toxin,  and  producing  coagulation-necrosis. 
Whatever  pyogenic  germs  happen  to  be  present  are  thus  afforded 
an  opportunity  to  enter  the  tissues  and  add  suppuration,  gangrene, 
and  remote  metastatic  lesions  to  the  already  existing  ulceration. 

Diphtheritic  inflammations  of  the  throat  are  not  always  accom- 
panied by  the  formation  of  the  pseudo-membrane,  but  in  some 
cases  a  rapid  inflammatory  edema  in  the  larynx,  without  a  fibrinous 
surface  coating,  may  cause  fatal  suffocation,  only  a  bacteriologic 
examination  revealing  the  true  nature  of  the  disease. 

Lesions. — The  pseudo-membrane  characterizing  diphtheria  con- 
sists of  a  combined  necrosis  of  the  tissues  acted  upon  by  the  toxin 
and  coagulation  of  an  inflammatory  exudate.  When  examined 
histologically  it  is  found  that  the  surface  of  the  mucous  membrane 
is  chiefly  affected.  The  superficial  layers  of  cells  are  emb'edded 
in  coagulated  exudate — fibrin — and  show  a  peculiar  hyaline  degenera- 
tion. Sometimes  the  membrane  seems  to  consist  exclusively  of 
hyaline  cells;  sometimes  the  fibrin  formation  is  secondary  to  or 
subsequent  to  the  hyaline  degeneration.  Leukocytes  caught  in 
the  fibrin  also  become  hyaline.  From  the  superficial  layer  the  process 
may  descend  to  the  deepest  layers,  all  of  the  cells  being  included 
in  the  coagulated  fibrin  and  showing  more  or  less  hyaline  degenera- 
tion. The  walls  of  the  neighboring  capillaries  also  become  hyaline, 
and  the  necrotic  mass  forms  the  diphtheritic  membrane.  The 
laminated  appearance  of  the  membrane  probably  depends  upon  the 
varying  depths  affected  at  different  periods,  or  upon  differences  in 
the  process  by  which  it  has  been  formed.  The  pseudo-membrane 
is  continuous  with  the  subjacent  tissues  by  a  fibrinous  reticulum, 
and  is  in  consequence  removed  with  difficulty,  leaving  an  abraded 
surface.  When  the  membrane  is  divulsed  during  the  course  of  the 
disease,  it  immediately  forms  anew  by  the  coagulation  of  the  in- 
flammatory exudate. 

The  coagulation-necrosis  seems  to  depend  upon  the  local  effect 
of  the  toxin.  Morax  and  Elmassian*  found  that  when  strong 
diphtheria  toxin  is  applied  to  the  conjunctiva  of  rabbits  every  three 
minutes  for  eight  or  ten  hours,  typical  diphtheritic  changes  are 
produced. 

*  "Ann.  de  1'Inst.  Pasteur,"  1898,  p.  210. 


Specificity  423 

Flexner*  has  made  a  study  of  the  minute  lesions  caused  by 
bacterial  toxins  and  especially  of  the  diphtheria  toxin,  and  Council- 
man, Mallory,  and  Pearce,f  of  both  gross  and  minute  lesions,  that 
the  thorough  student  should  read. 

Specificity. — Herman  Biggs, J  in  an  interesting  discussion  of  the 
occurrence  of  the  diphtheria  bacillus  and  its  relation  to  diphtheria, 
came  to  the  following  conclusions: 

1.  "When  the  diphtheria  bacillus  is  found  in  healthy  throats, 
investigation  almost  always  shows  that  the  individuals  have  been 
in  contact  with  cases  of  diphtheria.     The  presence  of  the  bacillus 
in  the  throat,  without  any  lesion,  does  not,  of  course,  indicate  the 
existence  of  the  disease." 

2.  "The  simple  anginas  in  which  virulent  diphtheria  bacilli  are 
found  are  to  be  regarded  from  a  sanitary  standpoint  in  exactly 
the  same  way  as  the  cases  of  true  diphtheria." 

3.  "Cases  of  diphtheria  present  the  ordinary  clinical  features  of 
diphtheria,  and  show  the  Klebs-Loffler  bacilli." 

4.  "Cases  of  angina  associated  with  the  production  of  membrane 
in  which  no  diphtheria  bacilli  are  found  might  be  regarded  from  a 
clinical  standpoint  as  diphtheria,  but  bacteriological  examination 
shows  that  some  other  organism  than  the  Klebs-Loffler  bacillus  is 
the  cause  of  the  process." 

Any  skepticism  of  the  specificity  of  the  diphtheria  bacillus  on 
my  own  part  was  dispelled  by  a  somewhat  unique  experience. 
Without  having  been  previously  exposed  to  diphtheria  while  ex- 
perimenting in  the  laboratory  the  author  accidentally  drew  a  living 
virulent  culture  of  the  diphtheria  bacillus  through  a  pipet  into  his 
mouth.  Through  carelessness  no  precautions  were  taken  to  prevent 
serious  consequences  and  two  days  later  the  throat  was  filled  with 
typical  pseudo-membrane  which  private  and  Health  Board  bacterio- 
logic  examinations  showed  to  contain  pure  cultures  of  the  Klebs- 
Loffler  bacilli. 

Some  have  been  led  to  doubt  the  specificity  of  the  diphtheria 
bacillus  because  of  the  existence  of  what  is  called  the  pseudo-diph- 
theria bacillus  or  bacillus  of  Hofmann  (q.v.).  Bomstein§  found 
that  though  it  was  possible  to  modify  the  activity  of  virulent 
bacilli,  and  bring  back  the  virulence  of  non-virulent  diphtheria 
bacilli,  it  was  impossible  to  make  the  pseudo-diphtheria  bacillus 
virulent.  Denny  ||  also  found  that  the  morphology  of  the  two  organ- 
isms- was  continually  different  when  they  were  grown  upon  the 
same  medium  for  the  same  length  of  time,  and  that  the  short  pseudo- 
diphtheria  bacillus  never  showed  any  tendency  to  develop  into  the 

*"  Johns  Hopkins  Hospital  Reports,"  vi,  259. 

t  "Diphtheria:  A  Study  of  the  Bacteriology  and  Pathology  of  Two  Hundred 
and  Twenty  Fatal  Cases,"  1901. 

t  "  Amer.  Jour.  Med.  Sci.,"  Oct.,  1896,  vol.  xxn,  No.  4,  p.  411. 
§  "Archiv  Russes  de  Path.,"  etc.,  Aug.  31,  1902. 
I!  American  Public  Health  Association,  1902. 


424  Diphtheria 

large  clubbed  forms  characteristic  of  the  true  diphtheria  organism. 
The  chief  points  of  difference  between  the  bacilli  are  that  the  pseudo- 
diphtheria  bacillus,  when  grown  upon  blood-serum,  is  short  and 
stains  uniformly;  that  cultures  grown  in  bouillon  develop  more 
rapidly  at  a  temperature  of  from  20°  to  22°C.  than  those  of  the 
true  bacillus;  and  that  the  pseudo-bacillus  is  not  pathogenic  for 
animals. 

Contagion. — The  diphtheria  bacilli,  being  always  present  in  the 
throats  of  patients  suffering  from  diphtheria,  constitute  the  element 
of  contagion. 

The  results  obtained  by  Biggs,  Park,  and  Beebe  in  New  York 
are  of  great  interest.  Bacteriologic  examinations  conducted  in 
connection  with  the  Health  Department  of  New  York  City  show 
that  virulent  diphtheria  bacilli  may  be  found  in  the  throats  of 
convalescents  from  diphtheria  as  long  as  five  weeks  after  the  dis- 
charge of  the  membrane  and  the  commencement  of  recovery,  and 
that  they  exist  not  only  in  the  throats  of  the  patients  themselves, 
but  also  in  those  of  their  caretakers,  who,  while  not  themselves 
infected,  may  be  the  means  of  conveying  the  disease  germs  from 
the  sick-room  to  the  outer  world.  Still  more  extraordinary  are  the 
observations  of  Hewlett  and  Nolen,*  that  the  bacilli  remained  in 
the  throats  of  patients  seven,  nine,  and  in  one  case  twenty-three 
weeks  after  convalescence.  The  hygienic  importance  of  this  ob- 
servation must  be  apparent  to  all  readers,  and  serves  as  further 
evidence  why  thorough  isolation  should  be  practised  in  connection 
with  the  disease. 

Neumann!  found  that  virulent  diphtheria  bacilli  may  occur  in 
the  nose  with  the  production  of  what  seems  to  be  a  simple  rhinitis 
as  well  as  a  pseudo-membranous  rhinitis.  Such  cases,  not  being 
segregated,  may  easily  serve  to  spread  the  contagion  of  the  disease. 

Wesbrook,  and  Wilson  and  McDanielJ  have  found  it  convenient 
to  describe  three  chief  types  of  the  diphtheria  bacillus  as  it  occurs 
in  twenty-four-hour-old  cultures  on  LofHer's  blood-serum,  sent  to 
the  laboratory  for  diagnosis.  The  classification  places  all  types  in 
three  general  groups:  (a)  granular,  (b)  barred,  and  (c)  solid  or 
evenly  staining  forms.  Each  group  is  subdivided  into  types  based 
on  the  shape  and  size  of  the  bacilli.  A  study  of  variations  in  the 
sequence  of  types  in  series  of  cultures  derived  from  clinical  cases  of 
diphtheria  shows  that  (a)  granular  types  are  usually  the  most 
predominant  forms  at  the  outset  of  the  disease;  (b)  the  granular  types 
usually  give  place  wholly  or  in  part  to  barred  and  solid  types  shortly 
before  the  disappearance  of  diphtheria-like  organisms;  (c)  solid  types, 
by  many  observers  called  "pseudo-diphtheria  bacilli,"  may  cause 
severe  clinical  diphtheria.  Solid  types  may  sometimes  be  re- 

*  "Brit.  Med.  Jour.,"  Feb.  i,  1896. 

f  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Jan.  24,  1902,  Bd.  xxxi,  No.  2,  p.  41. 

j  "Trans.  Assoc.  Amer.  Phys.,"  1900;  Trans.  Amer.  Public  Health  Asso.,  1900. 


Bacteriologic  Diagnosis 


425 


placed  by  granular  types  when  convalescence  is  established  and  just 
before  the  throat  is  cleared  of  diphtheria-like  bacilli. 

From  these  data  the  writers  conclude  that  it  is  not  safe  to  base 
an  opinion  regarding  the  maintenance  of  quarantine  upon  the 
bacterioscopic  findings  independently  of  the  clinical  history  of  the 
case. 

The  occurrence  of  true  diphtheria  bacilli  in  the  throats  of  healthy 
persons  has  been  a  stumbling-block  to  many  practitioners  unin- 
formed upon  bacteriologic  subjects,  who  fail  to  account  for  its 
presence  and  also  fail  to  realize  how  rare  its  appearance  under  such 
circumstances  really  is. 

Park*  found  virulent  diphtheria  bacilli  in  about  i  per  cent,  of 
the  healthy  throats  examined  in  New  York  city,  but  diphtheria 
was  prevalent  in  the  city  at  the  time,  and  no  doubt  most  of  the 


If    jfctlli 
—  I 


V, 


w 

Fig.  158. — Wesbrook's  types  of  Bacillus  diphtheriae:  a,  c,  d,  Granular  types; 
a1,  c1,  d1,  barred  types;  a2,  c2,  d2,  solid  types.     X  1500. 

persons  in  whose  throats  they  existed  had  been  in  contact  with 
cases  of  diphtheria.  He  very  properly  concludes  that  the  members 
of  a  household  in  which  a  case  of  diphtheria  exists,  though  they  have 
not  the  disease,  should  be  regarded  as  possible  sources  of  danger, 
until  cultures  made  from  their  throats  show  that  the  bacilli  have 
disappeared. 

Bacteriologic  Diagnosis. — It  is  impossible  to  make  an  accu- 
rate diagnosis  of  diphtheria  without  a  bacteriologic  examination. 

Such  an  examination  is  now  within  the  power  of  every  physician. 
All  that  is  required  is  a  swab  made  by  wrapping  a  little  absorbent 
cotton  about  the  end  of  a  piece  of  wire  and  carefully  sterilizing  it 
in  a  test-tube,  and  a  tube  of  Loffler's  blood-serum-medium,  that  can 

*  "  Report  on  Bacteriological  Investigations  and  Diagnosis  of  Diphtheria, 
from  May  4,  1893,  to  May  4,  1894."  "Scientific  Bulletin  No.  i,"  Health  De- 
partment, city  of  New  York. 


426 


Diphtheria 


be  bought  from  almost  any  modern  druggist.  The  swab  is  intro- 
duced into  the  throat  and  applied  to  the  false  membrane,  after 
which  it  is  carefully  smeared  over  the  surface  of  the  blood-serum. 
The  tube  thus  inoculated  is  stood  away  in  an  incubating  oven  or 
otherwise  kept  at  the  temperature  of  37°C.  for  twelve  hours,  then 
examined.  If  the  diphtheria  bacillus  be  present,  a  smeary,  creamy- 
white  layer  with  outlying  colonies  will  be  present.  These  colonies, 
if  found  by  microscopic  examination  to  be  made  up  of  diphtheria 

bacilli,  will  confirm  the  diag- 
nosis of  diphtheria.  There 
are  very  few  other  bacilli 
that  grow  so  rapidly  upon 
LofHer's  mixture,  and 
scarcely  any  other  is  found 
in  the  throat. 

When  no  tubes  of  the 
blood-serum  mixture  are  at 
hand,  the  swab  can  be  re- 
turned to  its  tube  after  hav- 
ing been  wiped  over  the 
throat  of  the  patient,  and  can 
be  shipped  to  the  nearest 
laboratory. 

When  an  early  diagnosis 
is  required,  Ohlmacher  rec- 
ommends that  the  micro- 
scopic examination  of  the 
still  invisible  growth  be  made 
in  five  hours.  A  platinum 
loop  is  rubbed  over  the  in- 
oculated surface;  the  small 
amount  of  material  thus  se- 
cured is  mixed  with  distilled 
water,  spread  on  a  cover- 
glass,  dried,  fixed,  stained 
with  methylene  blue,  and  ex- 
amined. An  abundance  of 
the  organisms  is  usually  found  and  valuable  time  is  saved  pre- 
paratory to  the  use  of  the  antitoxin. 

Diphtheria  Antitoxin. — Behring*  discovered  that  the  blood  of 
animals  rendered  immune  against  diphtheria  by  inoculation,  first 
with  attenuated  and  then  with  virulent  organisms,  contained  a 
neutralizing  substance  (Anti-korper)  capable  of  annulling  the  effects 
of  the  bacilli  or  the  toxin  when  simultaneously  or  subsequently 
inoculated  into  susceptible  animals.  This  substance,  held  in  solu- 

*"  Deutsche  med.  Wochenschrift,"  1890,  Nos.  49  and  50;  "Zeitschrift  fiir 
Hygiene,"  1892,  xn,  i. 


Fig.  159.— The  Providence  Health  De- 
partment outfit  for  diphtheria  diagnosis, 
consisting  of  a  pasteboard  box  containing 
a  swab-tube  and  a  serum-tube,  both  with 
etched  surface  on  which  to  write  the  name 
and  address  of  patients,  etc. 


Diphtheria  Antitoxin 


427 


tion  in  the  blood-serum  of  the  immunized  animals,  is  the  diphtheria 
antitoxin.  For  the  method  of  preparing  see  Antitoxins.  The  serum 
may  be  employed  for  purposes  of  prophylaxis  or  for  treatment. 

Prophylaxis. — The  serum  can  be  relied  upon  for  prophylaxis  in 
cases  of  exposure  to  diphtheria  infection.     In  most  cases  a  single 
dose  of  1000  units  is  sufficient  for  the  purpose.     The  protection  thus 
afforded   does   not   continue   longer   than 
about  six   weeks.     The   transitory  nature 
of  the  immunity  afforded  by  prophylactic 
injections    of    antitoxin   is   probably   de- 
pendent upon  the  fact  that  the  antitoxin  is 
slowly  eliminated. 

Treatment. — Diphtheria  antitoxin  is  al- 
ways to  be  administered  by  the  hypo- 
dermic method  at  some  point  where  the 
skin  is  loose.  Some  clinicians  prefer  to 
inject  into  the  abdominal  wall;  some,  into 
the  tissues  of  the  back.  A  slightly  painful 
swelling  is  formed,  which  usually  disap- 
pears in  a  short  time.  In  a  few  cases  sud- 
den death,  with  symptoms  suggesting  ana- 
phylaxis  (q.v.),  has  followed  the  injection. 

Ehrlich  asserts  that  a  dose  of  500  units 
is  valueless  for  the  treatment  of  diphtheria, 
2000  units  being  probably  an  average  dose 
for  an  adult  and  1000  units  for  a  child.  It 
is  far  better  to  err  on  the  side  of  administer- 
ing too  much  than  on  that  of  not  enough. 
Forty  thousand  units  have  been  adminis- 
tered to  a  moribund  child  with  resulting 
cure.  The  administration  of  the  remedy 
should  be  repeated  in  twelve  hours  if  the 
disease  is  one  or  two  days  old,  in  six  hours 
if  three  or  four  days  old,  in  four  hours 
if  still  older.  The  serum  may  have  to  be 
given  two,  three,  four,  or  even  more  times, 
according  to  the  case.  Occasionally  there 
is  an  outbreak  of  local  urticaria — rarely 

general  urticaria.  Sometimes  considerable  local  erythema  results. 
Fever  and  pain  in  the  joints  (serum  disease  of  von  Pirquet)  also 
occur,  especially  if  the  patients  have  been  previously  treated  with 
horse-serum. 

Diphtheria  paralysis  is  said  to  be  more  frequent  after  the  use  of 

antitoxin  than  in  cases  treated  without  it.     McFarland*  has  shown 

that  this  is  to  be  expected,  as  the  palsies  usually  occur  after  bad  cases 

of  the  disease,  of  which  a  far  greater  number  recover  when  antitoxin 

*"  Medical  Record,"  New  York,  1897. 


Fig.  1 60. — Sterilized 
test-tube  and  swab  for 
collecting  pus  and  fluids 
for  bacteriologic  examina- 
tion (Warren). 


428  Diphtheria 

is  used  for  treatment.  The  subject  has  been  worked  over  in  an  in- 
teresting manner,  from  the  experimental  side,  by  Rosenau.* 

An  interesting  collection  of  statistics  upon  the  antitoxic  treatment 
of  diphtheria  in  the  hospitals  of  the  world  has  been  published  by 
Professor  Welch, f  who,  excluding  every  possible  error  in  the  calcu- 
lations, "shows  an  apparent  reduction  of  case-mortality  of  55.8  per 
cent." 

Nothing  should  so  impress  the  clinician  as  the  necessity  of  begin- 
ning the  antitoxin  treatment  early  in  the  disease.  Welch's  statistics 
show  that  1115  cases  of  diphtheria  treated  in  the  first  three  days  of 
the  disease  yielded  a  fatality  of  8.5  per  cent.,  whereas  546  cases  in 
which  the  antitoxin  was  first  injected  after  the  third  day  of  the  dis- 
ease yielded  a  fatality  of  27.8  per  cent. 

On  the  other  hand,  it  can  scarcely  be  said  that  any  time  is  too  late 
to  begin  the  serum  treatment,  for  the  experiences  of  Burroughs  and 
McCollum  in  the  Boston  City  Hospital  show  that  by  immediate 
and  repeated  administration  of  very  large  doses  of  the  serum,  ap- 
parently hopeless  cases  far  advanced  in  the  disease,  may  often  be 
saved. 

After  the  toxin  has  occasioned  destructive  organic  lesions  of  the 
nervous  system  and  in  the  various  organs  and  tissues  of  the  body, 
no  amount  of  neutralization  can  restore  the  integrity  of  the  parts,  and 
in  such  cases  antitoxin  must  fail. 

One  disadvantage  under  which  the  diphtheria  antitoxic  serum  is 
administered  both  for  purposes  of  prophylaxis  and  treatment,  is 
the  inability  of  the  operator  to  find  out  what  may  be  the  already 
existing  antitoxin  content  of  the  patient's  blood.  Though  it  is  cer- 
tain that  existing  diphtheria  is  proof  that  the  patient  needs  the 
remedy,  it  is  by  no  means  certain  that  all  normal  persons  exposed 
to  diphtheria  in  institutions,  etc.,  require  it  for  prophylactic  purposes. 
Some  may  already  possess  enough  to  defend  them  and  the  promiscu- 
ous administration  of  the  serum  to  every  child  in  an  asylum,  may  re- 
sult in  sensitizing  some  to  the  allergizing  effect  of  the  horse-serum 
without  just  reason.  A  means  by  which  some  knowledge  of  the  nor- 
mal diphtheria-toxin  neutralizing  quality  of  the  blood  of  a  healthy 
individual  can  be  arrived  at,  has  been  devised  by  Schick,  {  and  is  now 
known  as  Schick's  reaction.  It  consists  in  the  intracutaneous  ad- 
ministration of  a  minute  dose  of  diphtheria  toxin.  If  the  patient's 
blood  contains  the  neutralizing  substance,  no  reaction  takes  place; 
if  it  contain  none,  a  reddened  and  tumefied  circumscribed  area  ap- 
pears. W.  H.  Park  uses  one-fiftieth  of  the  L+  dose  of  diphtheria 
toxin,  injecting  it  into  the  skin  with  a  very  fine  hypodermic  needle. 
Kolmer  prefers  to  use  one-fortieth  of  theL+  dose.  The  presence  of 
one-thirtieth  of  a  unit  of  antitoxin  in  i  cc.  of  the  patient's  blood  pre- 

*  "  Bulletin  No.  38  of  the  Hygienic  Laboratory,  U.  S.  Public  Health  and  Marine 
Hospital  Service,"  Washington,  D.  C.,  1907. 

f  "Bull,  of  the  Johns  Hopkins  Hospital,"  July  and  Aug.,  1895. 
j  "Miinchener.  med.  Wochenschrift,  1913,  p.  2605. 


Bacilli  Resembling  the  Diphtheria  Bacillus  429 

vents  the  reaction.  Kolmer*  has  also  made  use  of  the  Schick  reac- 
tion for  the  important  purpose  of  determining  how  long  the  anti- 
toxin serum  injected  into  the  patient  remains  and  confers  immunity. 
When  the  reaction  reappears,  the  immunity  can  be  supposed  to 
have  disappeared,  and  the  patient  again  becomes  susceptible  to  the 
infection. 

A  very  interesting  paper  by  Parkf  shows  the  effect  of  the  intro- 
duction of  antitoxin  upon  the  death-rate  from  diphtheria  and  the 
advantages  of  its  employment.  From  it  the  following  table  is  taken : 

"Combined  statistics  of  deaths  and  death-rates  from  diphtheria  and  croup  in 
New  York,  Brooklyn,  Boston,  Pittsburgh,  Philadelphia,  Berlin,  Cologne,  Bres- 
lau,  Dresden,  Hamburg,  Konigsberg,  Munich,  Vienna,  London,  Liverpool, 
Glasgow,  Paris,  and  Frankfort: 

Year  Peculation  Deaths  from  diph-  Deaths  per 

theria  and  croup  100,000 

1890 16,526,135  11,059  66.9 

1891 17,689,146  12,389  70.0 

1892 18,330,787          14,200          77 . 5 

1893 18,467,970         15,726        80.4 

1894 19,033,902        15,125       79.9 

1895! 19,143,188  10,657  55-6 

1896 19,489,682  9,651  49-5 

1897 19,800,629  8,942  45 . 2 

1898 20,037,918  7,170  35.7 

1899 20,358,837  7,256  35-6 

1900 20,764,614  6,791  32.7 

1901 20,874,572  6,104  29.2 

1902 21,552,398  5,630  26 .  i 

1903 21,865,299  5,117  23.4 

1904 22,532,848  4,917  21.8 

1905 22,790,000  4,323  19 .  o 

BACILLI  RESEMBLING  THE  DIPHTHERIA  BACILLUS 
BACILLUS  HOFMANNI 

The  pseudo-diphtheria  bacillus  (bacillus  of  Hofmann-Wellenhof  §) — 
Bacillus  pseudo-diphthericus — was  first  found  by  Lo frier ||  in  diph- 
theria pseudo-membranes  and  in  the  healthy  mouth  and  pharynx. 
Ohlmacher  has  found  it  with  other  bacteria  in  pneumonia;  Babes, 
in  gangrene  of  the  lung;  and  Ho  ward,**  in  a  case  of  ulcerative  endo- 
carditis not  secondary  to  diphtheria. 

Parkff  found  that  all  bacilli  with  the  typical  morphology  of  the 
diphtheria  bacillus,  found  in  the  human  throat,  are  virulent  Klebs- 
Loffler  bacilli,  while  forms  closely  resembling  them,  but  more 
uniform  in  size  and  shape,  shorter  in  length,  and  of  more  homo- 
geneous .  staining  properties  with  Loffler's  alkaline  methylene- 
blue  solution,  can  with  reasonable  safety  be  regarded  as  pseudo- 
diphtheria  bacilli,  especially  if  it  be  found  that  they  produce  an  alka- 

*"Phila.  Pathological  Society,"  Feb.  n,  1915. 

t  "Journal  of  the  Amer.  Med.  Assoc.,"  Feb.  17,  1912,  LVIII,  No.  7,  p.  453. 

t  Introduction  of  antitoxin  treatment. 

"Wiener  klin.  Woch.,"  1888,  No.  3. 

"Centralbl.  f.  Bakt.  u.  Parasitenk.,"  n,  105. 

"Bull,  of  the  Johns  Hopkins  Hospital,"  1893,  30. 
ft  "Scientific  Bulletin  No.  i,"  Health  Department,  city  of  New  York,  1895. 


43° 


Diphtheria 


line  rather  than  an  acid  reaction  by  their  growth  in  bouillon.  The 
pseudo-diphtheria  bacilli  were  found  in  about  i  per  cent,  of  throats 
examined  in  New  York ;  they  seem  to  have  no  relationship  to  diph- 
theria, and  are  never  virulent. 

Morphology. — -This  micro-organism  bears  a  more  or  less  marked 
resemblance  to  Bacillus  diphtherias,  but  differs  in  certain  particulars 
that  usually  make  it  possible  to  recognize  and  identify  it.  It  is 
shorter  and  stouter  than  its  relative,  is  straight,  usually  slightly 
clubbed.  It  usually  stains  intensely,  and  commonly  shows  but  one 
unstained  transverse  band.  When  the  bacilli  are  short  and  have  a 
single  band,  they  may  resemble  cocci.  When  longer  they  may  show 
two  transverse  bands. 

There  are  no  flagella  and  no  spores. 


v- 


Fig.   161. — Pseudo-diphtheria  bacilli. 

Staining. — -The  organism  stains  intensely  and  more  uniformly 
than  Bacillus  diphtheriae.  When  colored  by  Neisser's  or  Roux's 
method,  no  metachromatic  end  bodies  can  be  denned. 

Cultivation. — -The  organism  is  usually  discovered  in  smears  made 
for  the  diagnosis  of  diphtheria,  and  sometimes  occasions  considerable 
confusion  through  its  cultural  similarities  and  morphologic  resem- 
blances to  Bacillus  diphtheriae.  It  grows  more  luxuriantly  upon  the 
ordinary  culture-media  than  B.  diphtheriae.  The  colonies  are  larger, 
less  transparent  and  whiter,  as  seen  upon  agar-agar.  In  bouillon 
there  is  more  marked  clouding  and  less  marked  pellicle  formation. 
Upon  Loffler's  blood-serum  the  cultures  are  too  much  alike  to  be 
easily  differentiated. 

G.  F.  Petri*  found  no  substances  in  nitrates  of  cultures  of  Hof- 
mann's  bacillus  capable  of  neutralizing  diphtheria  antitoxin;  he  also 
found  that  horses  immunized  with  large  quantities  of  nitrates  of  the 
*  "Jour,  of  Hygiene,"  April,  1905,  vol.  v,  No.  2,  p.  134. 


Bacilli  Resembling  the  Diphtheria  Bacillus  431 

Hof  mann  bacillus  did  not  produce  any  antitoxin  to  diphtheria  toxin. 
Eleven  different  cultures  were  studied  and  the  results  are  very 
important. 

Cobbett*  and  Knappf  show  that  there  is  a  chemicobiologic  differ- 
ence between  the  true  and  pseudo-diphtheria  bacilli,  in  that  the 
latter  does  not  ferment  dextrin  or  any  of  the  sugars  as  the  true 
bacillus  does. 

Chemistry. — The  chemical  peculiarities  of  the  culture  serve  to 
make  certain  that  Bacillus  hofmanni  is  an  independent  micro-or- 
ganism. Under  no  circumstances  does  it  produce  or  can  it  be  made 
to  produce  toxin.  Under  no  circumstances  can  it  be  made  to  produce 
acid  through  the  decomposition  of  sugars. 

Pathogenesis. — Dr.  Alice  HamiltonJ  carefully  studied  29  organ- 
isms, of  which  26  corresponded  fully  with  the  pseudo-diphtheria 
bacilli.  They  were  divisible  into  three  groups :  I,  Those  non-patho- 
genic for  guinea-pigs;  II,  those  that  produce  general  bacteremia  in 
guinea-pigs,  and  are  neutralized  by  treatment  with  the  serum  of  a 
rabbit  immunized  against  a  member  of  the  group;  III,  organisms 
which  form  gas  in  glucose  media,  produce  bacteremia  in  guinea-pigs, 
and  are  neutralized  neither  by  diphtheria  nor  by  pseudo-diphtheria 
antitoxin.  Some  of  the  organisms  of  the  second  group  are  also 
pathogenic  for  man.  Instead  of  regarding  the  pseudo-diphtheria 
bacillus  as  a  harmless  saprophyte,  Dr.  Hamilton  believes  it  an  im- 
portant organism  explaining  some  of  the  paradoxes  that  we  find  at 
hand.  Thus,  cases  of  supposed  diphtheria  irremediable  by  or  dele- 
teriously  affected  by  antitoxic  serum  may  depend  upon  one  of  these 
organisms.  It  is  also  probably  one  of  them  that  Councilman  found 
in  his  case  of  "general  infection  by  Bacillus  diphtheriae,"  and  that 
Howard  encountered  in  his  case  of  acute  ulcerative  endocarditis  with- 
out diphtheria,  from  the  valves  of  whose  heart  cultures  of  a  diph- 
theria-like organism  not  pathogenic  for  guinea-pigs  was  isolated. 

The  still  more  recent  and  comprehensive  work  of  Clark  §  shows  that 
no  kind  of  manipulation  is  capable  of  so  modifying  Bacillus  hofmanni 
as  to  make  its  identity  with  B.  diphtheriae  in  the  least  likely.  Clark 
is,  however,  willing  to  admit  the  probability  that  the  organisms  may 
.have  descended  from  a  common  stock. 

BACILLUS  XEROSIS 

This  bacillus  was  first  described  in  1884  by  Kutschbert  and 
Neisser,||  who  regarded  it  as  the  cause  of  xerosis  conjunctivae,  having 
found  it  upon  the  conjunctiva  in  that  disease.  It  has,  however,  been 
so  frequently  found  upon  the  normal  conjunctiva  that  it  can  no 
longer  be  looked  upon  as  pathogenic.  It  is  also  found  upon  other 
*  'Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1898,  xxm,  395. 


'Jour,  of  Med.  Research,"  1904,  xn  (N.  S.,  vol.  vn),  p.  475 
'Jour.  Infectious  Diseases,"  1904,  i,  p.  690. 
'Journal  of  Infectious  Diseases,"  vn,  1910,  335. 
'Deutsche  med.  Wochenschrift,"  1884,  Nos.  21,  24. 


432  Diphtheria 

mucous  membranes  than  the  conjunctiva;  thus,  Leber  found  it  in  the 
mouth,  the  pelvis  of  the  kidney,  and  in  intestinal  ulcers.  From  the 
investigations  of  Sattler,  Frankel  and  Franke,  Schleich,  Weeks, 
Fick,  Baumgarten,  and  others  it  appears  that  Bacillus  xerosis  is  a 
harmless  saprophyte  that  is  occasionally  found  upon  the  conjunctiva. 
Happening  to  be  found  in  xerosis  it  was  accorded  undue  distinction. 

Morphology. — It  resembles  Bacillus  diphtheriae  very  closely,  but 
is  probably  a  little  shorter.  The  ends  are  clubbed,  and  in  them  meta- 
chromatic  bodies  are  stained  by  Neisser's  and  Roux's  methods. 

There  is  no  motility;  there  are  no  flagella  and  no  spores. 

Cultivation. — -Upon  Loffler's  medium  and  other  media  commonly 
used  for  the  diagnosis  of  diphtheria,  the  organism  grows  with  so  close 
resemblance  to  the  Bacillus  diphtheriae  as  to  make  the  differentiation 
difficult.  Transplanted  to  other  media,  it  continues  to  resemble 
B.  diphtheriae. 

Chemistry. — The  organism  is  incapable  of  forming  any  toxin. 
It  ferments  sugars  like  Bacillus  diphtheriae,  with  the  exception  of 
saccharose,  which  B.  xerosis  ferments,  but  which  B.  diphtheriae 
cannot  ferment.  B.  xerosis  also  fails  to  ferment  dextrin,  which  B. 
diphtheria  ferments. 

These  sugar-decomposing  properties  form  the  most  reliable 
methods  of  differentiating  Bacillus  diphtheriae,  B.  hofmanni, 
and  B.  xerosis. 

Pathogenesis. — The  organism  is  not  pathogenic  for  man  and  is 
certainly  not  the  cause  of  xerosis.  It  is  not  toxicogenic  and  is  not 
known  to  be  pathogenic  for  any  animal. 


CHAPTER  XIII 
VINCENT'S  ANGINA 

VINCENT'S  angina  is  an  acute,  specific,  infectious,  pseudo-membran- 
ous form  of  pharyngitis  or  tonsillitis  characterized  by  the  formation  of 
a  soft  yellowish-green  exudate  upon  the  mucous  membranes,  which, 
when  removed,  leaves  a  bleeding  surface  which  becomes  an  ulcer. 
Sometimes  these  ulcers  are  superficial,  sometimes  they  are  deep, 
necrotic,  and  fetid.  There  is  considerable  pain  on  swallowing,  some 
fever,  and  some  prostration.  The  patient  not  infrequently  keeps  up 
and  about,  though  feeling  very  badly.  The  ulcerations  sometimes 
persist  for  several  months.  As  there  is  considerable  swelling  of  the 
glands  of  the  neck  and  as  the  pseudo-membrane  is  sometimes  quite 
distinct,  the  disease  is  apt  to  be  mistaken  for  diphtheria,  and  may  be 
differentiated  from  it  only  by  a  bacteriologic  examination.  When 
such  an  examination  is  made  two  apparently  different  micro-or- 
ganisms may  be  found.  The  first  is  the  Bacillus  fusiformis;  the  sec- 
ond, Spirochaeta  vincenti. 

BACILLUS  FUSIFORMIS  (BABES  (?)) 

In  1882  Miller*  described  a  fusiform  bacillus  that  occurred  in 
small  numbers  between  the  gums  and  the  teeth  and  in  cavities  in 
carious  teeth  in  the  human  mouth.  In  1884  Cornil  and  Babesf  also 
described  a  fusiform  bacillus  which  seems  to  be  somewhat  different, 
that  occurred  in  a  necrotic  exudation  from  a  pseudo-membranous — 
diphtheritic — pharyngitis  in  school  children.  Lammershirt,  Vincent, 
Nicolle,  Plaut,  and  others  observed  similar  cases.  Later  Lichtowitz 
and  Sabrazes  observed  great  numbers  of  fusiform  bacilli  in  the  pus 
of  a  maxillary  empyema.  Elders  and  Matzenauer  observed  similar 
organisms  in  noma.  Fusiform  bacilli  are,  therefore,  not  infrequently 
associated  with  necrotic  processes  of  various  kinds.  Similar  but 
not  identical  bacilli  were  found  by  Babes  in  the  gums  of  scorbutic 
patients. 

SPIROCHAETA  VINCENTI  (PLAUT- VINCENT) 

PlautJ  and  Vincent§  observed  that  in  the  ulcerative  and  necrotic 
pharyngitis  described,  together  with  the  fusiform  bacilli,  there  were 
varying  numbers  of  spiral  organisms.  These  were  'difficult  to  stain, 

*  "Micro-organisms  of  the  Human  Mouth."     Philadelphia,  1890. 

t  "Lcs  Bacteries,"  1884. 

j  "  Deutsche  med.  Wochenschrift,"  1894,  XLEX. 

§  "Ann.  de  1'Inst.  Pasteur,"  1896,  488. 

28  433 


434  Vincent's  Angina 

always  took  faint  but  uniform  coloring,  varied  in  length,  and  showed 
such  irregular  and  non-uniform  undulations  as  to  appear  more  ser- 
pentine than  " corkscrew-like."  They  seem  never  to  occur  without 
associated  fusiform  bacilli.  The  writers  believe  these  organisms  and 
not  the  bacilli  to  be  the  cause  of  the  angina,  but  the  relation  of  the 
organisms  to  one  another  and  to  the  morbid  conditions  with  which 
they  were  associated  was  a  point  long  under  debate,  since  none  of 
those  studying  either  organism  succeeded  in  artificially  cultivating  it. 

RELATION  OF  THE  ORGANISMS  TO  ONE  ANOTHER 

We  have,  in  Vincent's  angina,  to  do  with  two  micro-organisms  that 
occur  in  habitual  association.  Neither  was  found  to  be  cultivable 
by  the  earlier  writers.  The  spirochaeta  could  not  be  cultivated  by 
Vincent,  and  of  the  various  fusiform  bacilli,  one  found  by  Babes  in 
scurvy,  which  was  obviously  different  from  the  others,  was  alone  sus- 
ceptible of  cultivation.  Later,  however,  reports  were  made  of  the 
growth  of  the  organisms  in  mixed  cultures.  Still  later,  Veillon  and 
Zuber,  Ellermann,  Weaver,  and  Tunnicliff  were  able  to  secure  pure 
cultures  of  the  fusiform  bacillus.  Quite  a  number  of  writers  reached 
the  conclusion  that  the  organisms  were  not  different,  but  were  dif- 
ferent stages  of  the  same  organism.  Tunnicliff*  found  that  in  pure 
cultures  of  Bacillus  fusiformis,  after  forty-eight  hours,  spiral  organ- 
isms resembling  those  seen  in  smear  preparations  from  the  original 
source  were  found.  From  Tunnicliff's  results  it  would  seem  as 
though  Bacillus  fusiformis  and  Spirochaeta  vincenti  are  identical 
organisms  in  different  stages  of  their  life-history.  But  the  matter 
is  not  yet  settled  for  Krumweide  and  Pratt*  by  a  different  method  of 
cultivation  have  apparently  obtained  Bufusiformis  pure — i.e.,  free 
from  the  spirochaeta — have  not  found  any  apparent  transformation 
of  the  bacilli  into  spirochaeta,  and  insist  that  the  two  are  essentially 
different  organisms. 

Cultivation. — The  organisms  were  cultivated  by  Tunnicliff  upon 
the  surface  of  ascitic  fluid  agar-agar  (i  :  3)  under  strictly  anaerobic 
conditions  at  37°C.  After  two  or  three  days  the  fusiform  bacillus 
appeared  in  the  form  of  delicately  whitish  colonies,  0.5  to  2  mm.  in 
diameter,  resembling  colonies  of  streptococci.  By  transplanting 
these,  pure  cultures  of  Bacillus  fusiformis  were  obtained.  In  the 
transplantation  tubes  the  organism  again  grew  in  the  form  of  similar 
whitish  colonies,  a  flocculent  deposit  accumulating  at  the  bottom  of 
the  water  of  condensation. 

Loffler's  Blood-serum  Mixture. — After  twenty-four  to  forty- 
eight  hours  similar  colonies  appear  and  a  similar  flocculent  deposit 
collects  in  the  condensation  water. 

Rabbit's  Blood  Agar-agar. — The  growth  is  similar,  but  brownish  in 
color. 

*"Jour.  of  Infectious  Diseases,"  1913,  XIH,  199;  438. 


Relation  of  the  Organisms  to  One  Another  435 

Glycerin  Agar-agar. — 'No  growth. 

Glucose  Agar-agar  Stab. — A  delicate  whitish   growth  with  small 


Fig.  162. — Bacillus  fusiformis.  Pure  culture  grown  forty-eight  hours  anae- 
robically  on  LofBer's  blood-serum.  (Ruth  Tunnicliff  in  "Journal  of  Infectious 
Diseases.") 

lateral  prolongations  develops  along  the  path  of  the  wire  in  twenty- 
four  to  forty-eight  hours.     Some  gas  is  formed. 

Litmus  Milk. — In  forty-eight  hours  there  is  a  moderate  growth. 


Fig.  163. — Bacillus  fusiformis.  Pure  culture  grown  forty-eight  hours  anae- 
robically  in  the  fluid  of  condensation  of  LofBer's  blood-serum.  (Ruth  Tunnicliff 
in  "Journal  of  Infectious  Diseases.") 


The  litmus  becomes  decolorized.     There  is  no  coagulation, 
oxygen  is  admitted  the  medium  regains  its  lost  color. 

Potato. — No  growth. 

Bouillon  and  Dextrin-free  Bouillon. — No  growth. 


When 


436 


Vincent's  Angina 


Glucose-bouillon. — -No  growth  when  more  than  i  per  cent,  of  glucose 
is  present.     The  medium  is  clouded  with  some  sediment. 
From  all  of  the  cultures  a  somewhat  offensive  odor  is  given  off. 


Fig.   164. — Bacillus  fusiformis.     Pure  culture  grown  four  days  in  ascites  broth 
(Ruth  Tunnicliff  in  "Journal  of  Infectious  Diseases.") 

Morphology. — The  Bacillus  fusiformis  presents  the  same  appear- 
ances, no  matter  what  medium  it  grows  upon.  It  measures  3  to  10  ju 
in  length,  0.3  to  0.8  p  in  thickness.  The  greatest  diameter  is  at  the 


Fig.  165. — Bacillus  fusiformis.     Smear  from  gum  in   normal  mouth.     (Ruth 
Tunnicliff  in  "Journal  of  Infectious  Diseases.") 

center,  from  which  the  organisms  gradually  taper  to  blunt  or  pointed 
extremities. 

The  organisms  stain  with  Lofner's  alkaline  methylene  blue,  with 
diluted  carbol-fuchsin,  by  Gram's  method,  and  by  Giemsa's  method. 


Pathogenesis  437 

The  staining  is  intense,  but.  is  rarely  uniform,  the  substance  usually 
being  interrupted  by  vacuoles  or  fractures,  reminding  one  of  those 
seen  in  the  diphtheria  and  tubercle  bacilli.  The  organism  forms  en- 
dospores  sometimes  situated  at  the  center,  but  more  frequently  to- 
ward one  end.  In  twenty-four  to  forty-eight  hours  filaments  are 
seen.  These  are  of  the  same  diameter  throughout,  and  usually  con- 
tain deeply  staining  bodies,  sometimes  round,  oftener  in  bands. 
Most  of  the  filaments  are  made  up  of  strings  of  bacilli,  but  some 
stain  uniformly.  Tunnicliff  found  that  about  the  fourth  or  fifth 
day  the  spirals  made  their  appearance,  sometimes  in  enormous 
numbers.  As  a  rule,  they  stained  uniformly,  some  showed  the 
dark  bodies  seen  in  the  bacilli  and  filaments.  They  had  from  one 
to  twenty  turns,  which  were  not  uniform.  The  spirals  were  flexible, 
the  ends  pointed.  The  spirals  persisted  in  the  cultures,  at  times 
for  fifty-five  days. 

Neither  the  bacilli  nor  the  spirals  showed  any  progressive  move- 
ment, though  with  the  dark-field  illuminator  they  showed  a  slight 
vibratile  and  rotary  movement.  No  flagella  were  observed. 

Pathogenesis. — Pure  cultures  of  the  organisms  were  inoculated 
into  guinea-pigs  without  result.  As  in  Vincent's  angina  the  throat 
always  contains  staphylococci  and  streptococci,  and  not  infrequently 
diphtheria  bacilli,  it  is  thought  by  many  that  Bacillus  fusiformis  does 
not  initiate  the  morbid  process,  but  is  a  secondary  invader,  by  which 
simpler  inflammations  are  intensified  and  made  necrotic. 

This  seems  to  be  particularly  true  of  diphtheria,  and  may  account 
for  the  occurrence  of  noma,  in  which  gangrenous  condition  of  the 
mouth  and  genitals  the  organisms  have  been  found  in  great  numbers. 

Bacillus  fusiformis,  with  the  associated  spirals  are  not  confined  to 
Vincent's  angina,  but  are  found  in  a  variety  of  other  necrotic  and 
gangrenous  affections.  Vincent*  himself  found  them  in  all  cases  of 
hospital  gangrene;  Veillon  and  Zuber,f  found  them  in  certain  cases 
of  appendicitis;  Bernheim  and  PopischellJ  in  gangrenous  laryngitis; 
Silberschmidt§  in  fetid  brochitis;  Freejmuth  and  Petruschky,|| 
Seiner**  and  others  in  noma;  Wolbachjf  in  certain  chronic  ulcers  of 
the  legs  in  Gambia. 

The  complete  literature  of  the  subject  collected  by  Beitzke,  is 
published  in  the  Centralbl.  fur  Bakt.  u.  Parasitenk.  (Referata)  1904, 
xxxv,  p.  i. 

*  "Ann.  de  1'Inst.  Pasteur,  1896,  x,  488. 

f  "  Archiv.  de  med.  Exp.,"  1898,  p.  517. 

j  "  Jahresb.  fur  Kinderheilkunde,"  1898,  XLV. 

§  "Centralbl.  f.  Bakt.,  etc.,"  1901,  Orig.,  xxx,  159. 

"Deutsche  med.  Wochenschrift,"  1898,  p.  232. 

"Wiener  klin.  Wochenschrift,"  1899,  No  2. 
ft  "Journal  of  Medical  Research,"  1912-13  xxvn,  27. 


CHAPTER  XIV 
THRUSH 

OIDIUM  ALBICANS  (ROBIN) 

THRUSH,  Soor  (German),  Muguet  (French),  or  parasite  stomatitis 
is  an  affection  of  marasmatic  infants  and  adults  characterized  by  the 
occurrence  of  peculiar  whitish  patches  upon  an  inflamed  oral  mucous 
membrane.  The  white  of  the  patches  consists  of  material  that  is  not 
easily  removed,  but  which  when  detached  leaves  a  bleeding  surface 
upon  which  it  forms  again.  Upon  microscopic  examination  the  white 
substance  proves  to  be  composed  of  masses  of  mycelia  with  enlarged 
epithelial  cells  and  leukocytes.  The  affection  is  far  more  frequent 
in  children  than  in  adults.  It  seems  not  to  occur  among  healthy 
children,  but  among  those  suffering  from  marasmus,  and  particu- 
larly among  those  whose  mouths  have  already  become  sore  through 
neglect.  It  is  usually  confined  to  the  mouth,  but  may  spread  to  the 
pharynx,  to  the  larynx,  in  rare  cases  to  the  esophagus,  in  very  rare 
cases  to  the  stomach  and  intestines,  and  in  exceptional  cases,  both  in 
adults  and  children,  may  become  a  generalized  disease  through 
hematogenous  distribution,  and  be  attended  by  mycotic  inflamma- 
tory lesions  in  the  kidneys,  the  liver,  and  the  brain. 

The  specific  micro-organism  seems  to  have  been  discovered  in  1839 
by  Langenbeck*  and  Berg.f  Langenbeck  missed  the  significance  of 
the  organism  altogether,  for,  finding  it  in  a  case  of  typhoid  fever,  he 
conceived  it  to  be  the  cause  of  that  disease.  Berg,  on  the  other  hand, 
regarded  it  as  the  cause  of  the  thrush.  Robin J  furnished  the  first 
correct  description  of  the  organism  and  gave  it  its  name,  Oidium 
albicans.  Many  systematic  writers  have  exercised  themselves  con- 
cerning the  exact  place  in  the  botanical  system  in  which  the  organisms 
should  be  placed.  Thus,  Gruby  and  Heim  regarded  it  as  a  sporo- 
trichum;  Robin,  as  an  Oidium;  Quinquaud,  as  a  syringospora; 
Hallein  called  it  Stemphylium  polymorpha;  Grawitz,  as  Myco- 
derma  vini;  Plaut,  as  Monilia  Candida;  Guidi,  Ress,  Brebeck-Fischer, 
as  a  saccharomyces;  Laurent,  as  Dematium  albicans;  Linossier  and 
Roux,  as  a  mucor,  and  Alav,  Olsen,  and  Vuillemin,  as  Endomyces 
albicans.  The  matter  is  still  undecided  and  until  it  is  finally  agreed 
upon  it  seems  best  to  resort  to  the  original  name,  Oidium  albicans. 

Morphology. — 'The  organism  consists  of  elements  that  bear  a  close 
resemblance  to  yeast  cells  and  multiply  by  budding,  of  hyphae  and 

*  See  Kehrer,  "Ueber  den  Soorpilz,"  etc.,  Heidelberg,  1883. 
f  See  Behrend,  "Deutsche  med.  Wochenschrift,"  1890. 

|"Histoire  naturelle  des  vegetaux  parasites  qui  croissent  sur  1'homme  et 
sur  les.animaux  vivants,"  Paris,  1853. 

438 


Cultivation  439 

mycelial  threads  into  which  these  grow,  and  of  chlamydospores  and 
conidia. 

The  yeast-like  elements  measure  5  to  6  /*  in  length  and  4  ^  in 
breadth.  They  have  an  oval  form  and  cannot  be  distinguished  from 
yeast  cells.  The  mycelia  are  formed  by  elongation  of  these  elements, 
some  of  which  appear  slightly  elongate,  some  greatly  elongate  and 
slender  and  more  or  less  septate,  like  those  of  the  true  molds.  They 
are  refractile,  doubly  contoured,  and  contain  droplets,  vacuoles,  and 
granules.  In  the  interior  of  the  hyphae  conidia-like  organs  often 
appear,  and  chalmydospores  are  found.  The  latter  are  large,  oval, 
doubly  contoured,  highly  refracting,  and  have  been  seen  by  Plaut 
to  germinate. 

The  morphology  is,  however,  extremely  varied,  and  the  greatest 
differences  of  interpretation  have  been  expressed  regarding  the  dif- 
ferent elements. 


Fig.  166. — Oldium.     (Kolle  and  Wassermann.) 

Cultivation. — The  organism  grows  readily  in  artificial  media,  both 
with  and  without  free  access  of  oxygen.  An  acid  reaction  is  most 
appropriate. 

Colonies, — -The  superficial  colonies  upon  gelatin  plates  are  rounded, 
waxy,  and  coarsely  granular.  The  deep  colonies  are  irregular  in 
shape  and  show  feathery  processes  extending  into  the  medium.  The 
color  varies  according  to  the  composition  of  the  medium,  from  snow 
white  on  ordinary  gelatin  to  meat-red  on  beet-root  gelatin.  A  sour 
odor  is  given  off  from  the  cultures. 

Gelatin  Punctures. — 'Along  the  line  of  puncture  there  is  a  slow  forma- 
tion of  rounded,  feathery,  colorless  colonies,  not  unlike  those  shown 
by  many  molds.  The  gelatin  is  slowly  liquefied  only  when  it  con- 
tains sugar.  In  such  cultures  chlamydospores  are  abundant. 

Agar-agar. — 'Cultures  are  similar  to  those  in  gelatin. 

Bouillon. — The  organism  grows  only  at  the  bottom  of  the  tube  in 
the  form  of  yellowish-white  flocculi. 


44°  Thrush 

Potato. — Various  in  different  cases.     Often  floury. 

M ilk. — The  organism  grows  very  poorly  in  milk,  which  is  not  coagu- 
lated or  fermented. 

Fermentation. — The  organism  utilizes  dextrin,  mannite,  alcohol, 
lactose,  and  glycerin  without  fermentation. 
Saccharose  is  destroyed  without  invertin  forma- 
tion. Glucose,  levulose,  and  maltose  are  fer- 
mented very  slowly. 

Metabolic  Products. — In  addition  to  the  fer- 
ments that  act  upon  the  sugars,  etc.,  and  soften 
the  gelatin,  the  organism  forms  alcohol,  aldehyd, 
and  acetic  acid. 

Pathogenesis. — Animals  are  not  known  to 
suffer  from  spontaneous  infection.  Grawitz  was 
able  to  induce  thrush  in  puppies.  Stooss  in- 
oculated the  scarified  vaginas  of  rabbits  with 
mixed  cultures  of  pyogenic  cocci  and  oidium 
and  obtained  thrush  plaques.  The  oidium 
alone  was  unable  to  secure  a  foothold.  Doder- 
lein,  Grosset,  and  Stooss  all  succeeded  in  pro- 
ducing abscesses,  sometimes  by  subcutaneous 
injection  of  the  oidium,  but  usually  only  when  it 
was  combined  with  pus  cocci.  In  such  abscesses 
the  cocci  are  killed  off  by  phagocytes,  and  when 
cultures  are  made  only  the  oidium  grows. 

Plaut  points  out  that  this  is  exactly  the  reverse 
Fig.   167. — Oidium      f     i        ,  ./-  .  i  e    i 

albicans.    Culture  in    oi  what  happens  in  artificial  cultures  of  the  two 

gelatin  (Hansen).  organisms  where  the  cocci  outgrow  and  kill  off 
the  oidium. 

Intravenous  injection  sometimes  causes  generalized  oiidium  infec- 
tion, with  colonies  of  the  micro-organism  in  the  kidneys,  heart- 
muscle,  peritoneum,  liver,  spleen,  stomach,  and  intestines.  The 
central  nervous  system  may  also  show  small  foci  of  the  infection. 

Immunity. — Roger*  and  Noissettef  were  able  to  immunize  ani- 
mals against  oidium. 

*  "  Compt.-rendu  de  la  Societe  de  Biologic,"  Paris,  1896. 
t  "  These  de  Paris,"  1898. 


CHAPTER  XV 
WHOOPING-COUGH 

THE  BORDET-GENGOU  BACILLUS 

THE  subacute,  contagious,  undoubtedly  infectious  disease  of 
childhood,  characterized  by  periodic  attacks  of  spasmodic  cough  and 
laryngeal  spasm,  terminating  in  a  prolonged  crowing  inspiration 
and  frequently  followed  by  vomiting  and  prostration,  known  as 
pertussis,  or  whooping-cough,  "  Keuchhusten "  (German)  and 
"coqueleuch"  (French),  has  long  been  subject  to  bacteriologic 
investigation.  Deichler,  Kurloff,  Szemetzchenko,  Cohn,  Neumann, 
Ritter,  and  Afanassiew  have  all  written  upon  bacteria  which  they 
supposed  to  be  the  causal  factors  of  the  disease,  but  which  time  has 
consigned  to  oblivion.  Koplik*  and  Czaplewski  and  Henselj  de- 
scribed micro-organisms  that  for  some.years  attracted  attention 
and  caused  more  or  less  discussion  as  to  which  might  be  the  real 
excitant  of  the  disease  or  whether  they  were  identical  organisms. 
As  time  passed,  both  observations  lacked  sufficient  confirmation  to 
carry  conviction  of  their  importance,  and  they,  too,  fell  into  oblivion. 
A  still  different  organism  was  described  by  Vincenzi,J  but  also  failed 
to  meet  sufficient  confirmatory  evidence  to  prevent  it  from  meeting 
the  fate  of  its  predecessors. 

Spengler,§  Krausand  Jochmann,||  and  Davis**  showed  the  frequent 
presence  of  minute  bacilli  in  the  sputum  and  also  in  the  lesions  of  the 
disease.  They  were,  almost  beyond  doubt,  influenza  bacilli. 

In  1906  Bordet  and  Gengouft  described  a  new  organism  whose 
importance  was  supported  by  such  weighty  evidence  as  the  forma- 
tion of  an  endotoxin  sufficiently  active  to  explain  the  symptoms,  and 
the  fixation  of  complement  by  the  serum  of  the  infected  animal. 
This  organism,  therefore,  presents  itself  as  sufficiently  meritorious 
to  maintain  the  field  for  the  present. 

Morphology. — The  organisms,  as  found  in  the  sputum,  occur  as 
very  minute  ovoid  rods  of  about  the  same  size  as  the  influenza 
bacillus.  They  measure  approximately  i-5/x  in  length  by  0.3  n  in 
breadth.  They  do  not  remain  united  as  chains  or  rods,  but  separate 

*  "Centralbl.  f.  Bakt.,"  etc.,  Sept.  15,  1897,  xxn,  8  and  9,  p.  222. 
f'Deutsch.    med.    Wochenschrift,"    1897,   No.   57,  p.    586;   "Centralbl.   f. 

Bakt.,"  etc.,  Dec.  22,  1897,  xxn,  Nos.  22  and  23,  p.  641. 

t"Atti  della  Accademia  di  Medicina- in  Torino,"  LXI,  5-7;  "Centralbl.  f. 
Bakt.,"  etc.,  Jan.  19,  1898,  xxm,  p.  273. 

§  "Deutsch.  med.  Wochenschrift,"  1897,  830. 
"Zeitschrift  fur  Hygiene,"  etc.,  1901,  xxxvi,  193. 

*  "Jour.  Infectious  Diseases,"  1906,  in,  i. 
ft  "Ann.  de  1'Inst.  Pasteur,"  1906,  xx,  731. 

44i 


44 2  Whooping-cough 

as  individuals.  They  are  somewhat  pleomorphous,  yet  the  varia- 
tions are  not  considerable.  Involution  forms  are  not  common. 
There  are  no  spores,  no  flagella,  no  motility. 

Staining. — The  organisms  do  not  hold  the  stain  well.  Most  of 
the  bacilli  are  pale,  some  contain  uncolored  areas  or  vacuoles.  In 
some  cases  the  ends  of  the  bacilli  appear  more  deeply  stained  than 
the  middle.  They  do  not  stain  by  Gram's  method.  The  discoverers 
recommend  that  the  organism  be  stained  with — 


Toluidin  blue.. .  <  }  ,-,.      ,  , 

Alcohol ioo     Evolve  and  add  500  of  5  per  cent,  aqueous 

Water 500  J 


carbolic  acid.     After  two  days  filter. 


Isolation. — The  organisms  occur  in  almost  pure  cultures  in  the 
whitish  expectoration  which  escapes  from  the  bronchi  in  the  begin- 
ning of  the  disease.  Later  they  become  few  and  may  disappear, 
though  the  symptoms  of  the  disease  persist. 


Fig.  1  68.  —  The  Bordet-Gengou  bacillus  of   whooping-cough.     Twenty-four- 
hour-old  culture  upon  solid  media  containing  blood  (Bordet-Gengou). 

Cultivation.  —  The  cultures  were  secured  upon  a  special  medium 
made  as  follows: 


II.  Potato  extract  (made  as  above)  ..   50  cc.  )  Boil,     dissolve,      filter,      and 
0.6  per  cent,  aqueous  Nad  ......  150  cc.    f      tube;    2    to    3    cc.     to     a 

Agar-agar  ......................   5  gm.  J       tube. 

III.  To  each  tube  add  an  equal  volume  of  defibrinated  rabbits'  (or,  better, 
human)  blood  before  cooling  to  the  point  of  coagulation.  Permit  the  tubes 
to  solidify  in  the  oblique  position. 

At  first  the  growth  is  scant,  but  upon  transplantation  grows  better 
and  better,  until  finally  it  may  be  made  to  grow  upon  other  media, 
such  as  blood-agar,  ascitic  agar,  or  broth  to  which  blood  or  ascitic 
fluid  has  been  added.  The  organism  is  a  strict  aerobe.  It  grows 
best  at  37°C.,  but  also  grows  at  temperatures  as  low  as  5°  to  io°C. 


Pathogenesis  443 

On  appropriate  culture-media  Wollstein  found  it  might  remain  alive 
for  two  months. 

Metabolic  Products.— An  endotoxin  was  found  by  Bordet  and 
Gengou,  the  method  of  preparing  which  was  improved  by  Besredka* 
as  follows:  The  growth  upon  agar-agar  is  removed  with  a  small 
quantity  of  salt  solution,  dried  in  vacuo,  and  ground  in  a  mortar 
with  a  small  measured  quantity  of  salt.  Enough  distilled  water  is 
then  added  to  make  a  0.75  per  cent,  solution,  after  which  the  mixture 
is  centrifugalized  and  decanted.  Of  this  preparation  i  to  2  cc.  usu- 
ally killed  a  rabbit  about  twenty-four  hours  after  intravenous  injec- 
tion. Subcutaneous  injection  caused  a  necrosis  without  suppuration 
and  without  constitutional  symptoms.  Small  quantities  of  the  toxin 
placed  in  the  rabbit's  eye  caused  local  necrosis,  with  little  inflam- 
matory reaction.  The  introduction  of  dead  or  living  cultures  into 
the  peritoneal  cavity  of  guinea-pigs  caused  death  with  great  effusion 
and  hemorrhage  in  the  peritoneal  tissues. 

Pathogenesis. — Inoculation  of  monkeys  with  cultures  of  the  ba- 
cillus failed  to  produce  the  disease.  Klimenko,t  however,  succeeded 
in  infecting  monkeys  and  pups  by  intratracheal  introduction  of 
pure  cultures.  After  a  period  of  incubation  an  illness  came  on,  the 
most  marked  symptoms  being  pyrexia  and  pulmonary  irritation. 
After  two  or  three  weeks  the  dogs  died.  Postmortem  examination 
showed  catarrh  of  the  respiratory  tissues  with  patches  of  broncho- 
pneumonia.  Healthy  dogs  contracted  the  disease  by  contact  with 
those  suffering  from  the  infection.  Frankel |  obtained  similar  results. 

The  differences  between  the  Bordet-Gengou  bacillus  and  the  in- 
fluenza bacillus  are  not  great.  In  size,  mode  of  occurrence,  grouping 
and  staining  there  is  much  resemblance  between  the  two.  Cultur- 
ally, however,  they  differ  because  the  influenza  bacillus  grows  best 
upon  hemoglobin  or  blood  agar-agar,  which  is  less  adapted  for  the' 
isolation  of  the  Bordet-Gengou  bacillus  than  the  culture-medium 
recommended  for  its  cultivation,  upon  which  the  influenza  bacillus 
does  not  grow  well.  Further,  we  have  as  differential  features  the 
peculiar  endotoxin  of  the  Bordet-Gengou  bacillus,  the  successful 
infection  of  dogs  and  monkeys  with  the  disease  resembling  whoop- 
ing-cough, and  the  transmission  of  this  infection  from  animal  to 
animal  by  natural  means. 

The  subject  of  complement  deviation  as  a  proof  of  the  specific 
nature  of  the  organism  is  still  under  consideration.  Bordet  and 
Gengou  found  that  the  serum  of  convalescent  patients  fixed  com- 
plement when  applied  to  the  bacilli;  Frankel  and  Wollstein, §  that 
it  did  not.  It  is  claimed  by  Bordet  and  Gengou  that  the  difference 
in  results  came  about  through  the  employment  of  different  culture- 
media  in  performing  the  complement  fixation  tests. 

*  Bordet,  "Bull,  de  la  Soc.  Roy.  de  Bruxelles,"  1907. 
t"Centralbl.  f.  Bakt.,"  etc.  (Orig.),  XLVIII,  64. 
t  "Miinchener  med.  Wochenschrift,"  1908,  p.  1683. 
§  "Journal  of  Exp.  Med.,"  1909,  xi,  41. 


CHAPTER  XVI 

PNEUMONIA 
LOBAR  OR  CROUPOUS  PNEUMONIA 

DIPLOCOCCUS  PNEUMONIA  (WEICHSELBAUM) 

General  Characteristics. — A  minute,  spheric,  slightly  elongate  or  lancet- 
shaped,  non-motile,  non-flagellate,  non-sporogenous,  aerobic  and  optionally 
anaerobic,  non-chromogenic,  non-liquefying  diplococcus,  pathogenic  for  man  and 
he  low  er  animals,  staining  by  ordinary  methods  and  by  Gram's  method. 

"Pneumonia,"  while  generally  understood  to  refer  to  the  lobar 
form  of  the  disease  particularly  designated  as  croupous  pneumonia, 
is  a  vague  term,  comprehending  a  number  of  quite  dissimilar  in- 
flammatory conditions  of  the  lung.  This  being  true,  no  single 
micro-organism  can  be  " specific"  for  all.  Indeed,  pneumonia 
must  be  conceived  of  as  a  group  of  diseases,  and  the  various  micro- 
organisms associated  with  it  must  be  separately  considered  in  con- 
nection with  the  particular  varieties  of  the  disease  in  which  they 
occur. 

The  microorganism,  that  can  be  demonstrated  in  at  least  75  per 
cent,  of  cases  of  lobar  pneumonia,  which  is  almost  universally  ac- 
cepted to  be  the  cause  of  the  disease,  and  about  whose  specificity 
very  few  doubts  can  now  be  raised,  is  the  Diplococcus  pneumonias 
or  pneumococcus,  of  Frankel  and  Weichselbaum. 

Priority  of  discovery  of  the  pneumococcus  seems  to  be  in  favor 
of  Sternberg,*  who  as  early  as  1880  described  an  apparently  identical 
organism  which  he  secured  from  his  own  saliva.  Pasteurf  seems  to 
have  cultivated  the  same  micro-organism,  also  from  saliva,  in  the 
same  year.  The  researches  of  the  observers  whose  names  are  now 
attached  to  the  organism  were  not  completed  until  five  years  later. 
It  is  to  Telamon,t  Frankel,§  and  particularly  to  Weichselbaum, || 
however,  that  we  are  indebted  for  the  discovery  of  the  relation  which 
the  organism  bears  to  pneumonia. 

Distribution. — 'The  pneumococcus  is  one  of  a  group  of  widely  dis- 
seminated organisms  of  the  respiratory  tract.  It  is  characterized 
by  certain  peculiarities  of  morphology,  certain  metabolic  peculiari- 
ties, a  definite  pathogenesis,  and  a  distinct  agglutinative  reaction 

*  "National  Board  of  Health  Bulletin,"  1881,  vol.  n. 

f  "  Compte-rendus  Acad.  des  Sciences,"  1881,  xcn,  p.  159. 

j  "Compte-rendus  de  la  Societed'  anatom.  de  Paris,"  Nov.  30,  1883. 

§  "-Deutsche  med.  Wochenschrif t, "  1885,  31. 

||  "Wiener  med.  Jahrbuch,"  1886,  p.  483. 

444 


Staining  445 

with  immune  serum.  Recent  researches  make  it  certain  that  some 
of  the  organisms  formerly  looked  upon  as  pneumococci  are  different 
and  perhaps  harmless.  The  pneumococcus  is  a  purely  parasitic, 
pathogenic  organism,  best  known  to  us  in  croupous  pneumonia, 
where  it  is  present  in  the  lungs,  sputum,  and  blood.  It  may  be 
found  in  the  saliva  of  a  large  number  of  healthy  persons  (Parke 
and  Williams*),  especially  during  the  winter  months  (Longcope  and 
Foxf),  and  the  inoculation  of  human  saliva  into  rabbits  frequently 
causes  septicemia  in  which  the  pneumococci  are  abundant  in  the 
blood  and  tissues.  Its  frequent  occurrence  in  the  saliva  led  Fliigge 
to  describe  it  as  Bacillus  septicus  sputigenus.  It  is  occasionally 
found  in  inflammatory  lesions  other  than  pneumonia,  as  will  be 
pointed  out  below. 

Morphology. — The  organism  is  variable  in  morphology.  When 
grown  in  bouillon  it  appears  oval,  has  a  pronounced  disposition  to 
occur  in  pairs,  and  not  infrequently  forms  chains  of  five  or  six  mem- 
bers, so  that  some  have  been  disposed  to  look  upon  it  as  a  streptococ- 
cus (Gamale'ia) .  In  the  fibrinous  exudate  from  croupous  pneumonia, 
in  the  rusty  sputum,  and  in  the  blood  of  rabbits  and  mice,  the  organ- 
isms occur  in  pairs,  have  a  lanceolate  shape,  the  pointed  ends 
usually  being  approximated,  and  are  usually  surrounded  by  a  distinct 
halo  or  capsule  of  clear,  colorless,  homogeneous  material,  thought  by 
some  to  be  a  swollen  cell-wall,  by  others  a  mucus-like  secretion 
given  off  by  the  cells.  When  grown  in  culture-media,  especially 
upon  solid  media,  the  capsules  are  not  apparent.  The  elongate 
form  has  led  MigulaJ  to  describe  it  under  the  name  Bacterium 
pneumoniae. 

The  organism  measures  about  i  n  in  greatest  diameter,  is  without 
motility,  has  no  flagella  and  forms  no  spores. 

Staining. — -It  stains  well  with  the  ordinary  solutions  of  the  anilin 
dyes,  and  gives  most  beautiful  pictures  in  blood  and  tissues  when 
stained  by  Gram's  and  Weigert's  methods. 

To  demonstrate  the  capsules,  the  glacial  acetic  acid  method  of 
Welch§  may  be  used.  The  cover-glass  is  spread  with  a  thin  film  of 
the  material  to  be  examined,  which  is  dried  and  fixed  as  usual. 
Glacial  acetic  acid  is  dropped  upon  it  for  an  instant,  poured  (not 
washed)  off,  and  at  once  followed  by  anilin-water  gentian  violet,  in 
which  the  staining  continues  several  minutes,  the  stain  being  poured 
off  and  replaced  several  times  until  the  acid  has  all  been  removed. 

Finally,  the  preparation  is  washed  in  water  containing  i  or  2  per 
cent,  of  sodium  chlorid,  and  may  be  examined  at  once  in  the  salt 
solution,  or  mounted  in  balsam  after  drying.  The  capsules  are  more 
distinct  when  the  examination  is  made  in  water. 

*  "Jour.  Exp.  Med.,"  Aug.  7,  1905,  vn,  p.  403. 

t  Ibid.,  p.  430. 

J  "System  der  Bakterien,"  Jena,  1900,  p.  347. 

§  "Bull,  of  the  Johns  Hopkins  Hospital,"  Dec.,  1892,  p.  128.* 


446  Pneumonia 

Hiss*  recommends  the  following  as  an  excellent  method  of  stain- 
ing the  capsules  of  the  pneumococcus:  The  organism  is  first  culti- 
vated upon  ascites  serum-agar  to  which  i  per  cent,  of  glucose  is 
added.  The  drop  containing  the  bacteria  to  be  stained  is  spread 
upon  a  cover-glass  mixed  with  a  drop  of  serum  or  a  drop  of  the  fluid 
culture-medium,  and  dried  and  fixed.  A  half-saturated  aqueous 
solution  of  gentian  violet  is  applied  for  a  few  seconds  and  then  washed 
off  in  a  25  per  cent,  solution  of  carbonate  of  magnesium.  The 
preparation  is  then  mounted  in  a  drop  of  the  latter  solution  and 
examined. 

If  it  is  desired  to  stain  the  capsules  and  preserve  the  specimens 
permanently  in  balsam,  Hiss  employs  a  5  or  10  per  cent,  solution 
of  fuchsin  or  gentian  violet  (5  cc.  saturated  alcoholic  solution  of 
dye  in  95  cc.  of  distilled  water).  The  stain  is  applied  to  the  fixed 


Fig.  169. — Capsulated  pneumococci  in  blood  from  the  heart  of  a  rabbit;  carbol- 
fuchsin,  partly  decolorized.      X  1000. 

specimen  and  heated  until  it  begins  to  steam,  when  the  stain  is 
washed  off  in  a  20  per  cent,  solution  of  crystals  of  sulphate  of  copper. 
The  preparation  is  then  dried  and  mounted  in  balsam. 

Hiss  finds  this  stain  a  useful  aid  in  differentiating  the  pneumo- 
coccus from  the  streptococcus,  with  which  it  is  easily  confounded  if 
the  capsules  are  not  distinct,  and  to  which  it  is  probably  closely 
related. 

Isolation. — When  desired  for  purposes  of  study,  the  pneumococcus 
may  be  obtained  by  inoculating  white  mice  with  pneumonic  sputum 
and  recovering  the  organisms  from  the  heart's  blood,  or  it  may  be 
obtained  from  the  rusty  sputum  of  pneumonia  by  the  method  em- 
ployed by  Kitasato  for  securing  tubercle  bacilli  from  sputum:  A 
mouthful  of  fresh  sputum  is  washed  in  several  changes  of  sterile 

*  Abstract,  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Bd.  xxxi,  No.  10,  p.  302, 
March~24,  1902.  More  complete  details  appear  in  a  later  paper  in  the  "Journal 
of  Experimental  Medicine,"  vi,  p.  338. 


Cultivation 


447 


water  to  free  it  from  the  bacteria  of  the  mouth  and  pharynx,  care- 
fully separated,  and  a  minute  portion  from  the  center  transferred 
to  an  appropriate  culture-medium. 

Buerger,*  in  conducting  a  research  upon  pneumococcus  and  allied 
organisms  with  reference  to  their  occurrence  in  the  human  mouth, 
under  the  auspices  of  the  Rockefeller  Institute,  used  a  2  per  cent, 
glucose-agar  of  a  neutral,  or,  at  most,  0.5  per  cent,  phenolphthalein 
acid  titer. 

"The  medium  was  usually  made  from  meat  infusion  and  contained  1.5  to  2 
per  cent,  peptone  and  2.4  per  cent.  agar.  Stock  plates  of  these  media  (serum  - 
agar  and  2  per  cent,  glucose-serum-agar)  were  poured.  The  agar  or  glucose- 
agar  was  melted  in  large  tubes  and  allowed  to  cool  down  to  a  temperature  below 
the  coagulation  point  of  the  serum.  One-third  volume  of  rich  albuminous 
ascitic  fluid  was  added,  and  the  resulting  media  poured  into  Petri  plates.  These 
were  tested  by  incubation  and  stored  in  the  ice-chest  ready  for  use.  .  .  . 

"The  plan  finally  adopted  [for  inoculating  the  plates]  was  as  follows:  A 
swab  taken  from  the  mouth  was  thoroughly  shaken  in  a  tube  of  neutral  bouillon. 
From  this  primary  tube,  dilutions  in  bouillon  with  four,  six,  and  eight  loops 
may  be  made.  A  small  portion  of  the  dilute  mixture  was  poured  at  a  point  near 
the  periphery  of  the  prepared  plates.  By  a  slight  tilting  motion  the  fluid  was 
carefully  distributed  over  the  whole  surface  of  the  plates.  Care  must  be  taken 
to  avoid  an  excess  of  fluid.  It  was  found  that  plates  made  in  this  way  gave 
a  sufficiently  thick  and  discrete  distribution  of  surface  colonies." 

Cultivation. — The  organism  grows  upon  all  the  culture-media  ex- 
cept potato,  but  only  between  the  temperature  extremes  of  24°  and 
42°C.,  the  best  development  being  at  about  37°C.  The  growth  is 
always  meager,  probably  because  of  the  metabolic  formation  of 
formic  acid.  The  addition  of  alkali  to  the  culture-medium  favors 
the  growth  of  the  pneumococcus  by  neutralizing  this  acid.  Hiss  and 
Zinsserf  advise  that  the  culture-media  used  for  the  pneumococcus 
be  made  with  3  to  4  per  cent,  of  peptone. 

Colonies. — The  colonies  which  develop  at  24°C.  upon  gelatin 
plates  (15  per  cent,  of  gelatin  should  be  used  to  prevent  melting  at 
the  temperature  required)  are  described  as  small,  round,  circum- 
scribed, finely  granular  white  points  which  grow  slowly,  never  attain 
any  considerable  size,  and  do  not  liquefy  the  gelatin. 

If  agar-agar  be  used  instead  of  gelatin,  and  the  plates  kept  at  the 
temperature  of  the  body,  the  colonies  appear  transparent,  delicate, 
and  dewdrop-like,  scarcely  visible  to  the  naked  eye,  but  under  the 
microscope  appear  distinctly  granular,  a  dark  center  being  sur- 
rounded by  a  paler  marginal  zone. 

Upon  the  medium  recommended  by  Buerger  for  isolating  the 
pneumococcus,  the  colonies  appear  in  from  eighteen  to  twenty-four 
hours,  the  surface  colonies  being  circular  and  disk-like.  When 
viewed  from  above,  the  surface-  appears  glassy  with  a  depressed 
center.  When  viewed  from  the  side  or  by  transmitted  light,  they 
appear  as  distinct  milky  rings  with  a  transparent  center.  This 


*  "Jour.  Exp.  Med.,"  Aug.  25,  1905,  vn,  No.  5. 
t  "Text-book  of  Bacteriology,"  1910,  p. 


356. 


448  Pneumonia 

"ring  type"  is  regarded  as  characteristic  and  enables  the  organism 
to  be  separated  without  difficulty  from  the  streptococcus. 

Gelatin  Punctures. — In  gelatin  puncture  cultures,  made  with 
15  instead  of  the  usual  10  per  cent,  of  gelatin,  the  growth  takes 
place  along  the  entire  puncture  in  the  form  of  minute  whitish  gran- 
ules distinctly  separated  from  one  another.  The  growth  in  gelatin 
is  always  meager.  The  medium  is  not  liquefied. 

Agar-agar  and  Blood-serum. — Upon  agar-agar  and  blood-serum 
the  growth  consists  of  minute,  transparent,  semi-confluent,  colorless, 
dewdrop-like  colonies.  The  medium  is  not  liquefied.  Upon  glycerin 
agar-agar  the  growth  is  more  luxuriant.  The  addition  of  a  very 
small  percentage  of  blood-serum  facilitates  growth. 

Bouillon. — In  bouillon  the  organisms  grow  well,  slightly  clouding 
the  medium.  With  the  death  of  the  organisms  and  their  sedimenta- 
tion, the  medium  clears  again  after  a  few  days. 

Milk. — Milk  is  an  appropriate  culture-medium,  its  casein  being 
coagulated.  Alkaline  litmus  milk  is  slowly  acidified. 

Potato. — The  pneumococcus  does  not  grow  upon  potato.* 

Vital  Resistance. — The  organism  usually  dies  after  a  few  days  of 
artificial  cultivation,  and  so  must  be  transplanted  every  three  or 
four  days.  In  rabbit's  blood,  in  sealed  tubes  kept  cold,  it  can  some- 
times be  kept  alive  for  several  weeks.  Hiss  and  Zinsserf  find  that 
when  the  organism  is  planted  in  "calcium-carbonate-infusion  broth" 
and  kept  in  the  ice-chest,  the  cultures  often  remain  alive  for  several 
months.  Bordoni-UffreduzziJ  found  that  when  pneumococci  were 
dried  in  sputum  attached  to  clothing,  and  were  exposed  freely  to  the 
light  and  air,  they  retained  their  virulence  for  rabbits  for  from  nine- 
teen to  ninety-five  days.  Direct  sunlight  destroyed  their  virulence 
in  twelve  hours.  Guarniere§  found  that  dried  blood  containing 
pneumococci  remained  virulent  for  months. 

The  pneumococcus  is  destroyed  in  ten  minutes  by  a  temperature 
of  52°C.  It  is  highly  sensitive  to  all  disinfectants,  weak  solutions 
quickly  killing  it. 

Metabolic  Products. — Hiss||  found  that  the  pneumococcus  pro- 
duces acid  from  monosaccharids,  disaccharids,  and  such  complex 
saccharids  as  dextrin,  glycogen,  starch,  and  inulin.  The  fermenta- 
tion of  inulin  by  pneumococci  is  a  most  important  means  of 
differentiating  it  from  streptococci. 

Toxic  Products. — Nothing  definite  is  known  about  the  metabolic 
toxic  products  of  the  pneumococcus. 

Auld**  found  that  if  a  thin  layer  of  prepared  chalk  were  placed 

*  Ortmann  asserts  that  the  pneumococcus  can  be  grown  on  potato  at  37°C., 
but  this  is  not  generally  admitted.  The  usual  acid  reaction  of  potato  makes 
it  an  unsuitable  culture-medium. 

t  LOG.  cit. 

J"  Arch.  p.  1.  Sc.  Med.,"  1891,  xv. 
"Atti  della  R.  Acad.  Med.  di  Roma,"  1888,  iv. 
"Jour.  Exp.  Med.,"  vn,  No.  5,  Aug.  25,  1905. 
"Brit.  Med.  Jour.,"  Jan.  20,  1900. 


Pathogenesis  449 

upon  the  bottom  of  the  culture-glass,  it  neutralized  the  lactic  acid 
produced  by  the  pneumococcus,  and  enabled  it  to  grow  better  and 
produce  much  stronger  toxin.  Macfadyen*  found  that  by  freezing 
cultures  of  the  pneumococcus  with  liquid  air,  destroying  them 
by  trituration  in  the  frozen  state  and  then  extracting  the  frag- 
ments with  i  :  1000  caustic  potash  solution,  a  toxin  whose  activity 
corresponded  fairly  well  with  the  virulence  of  the  culture  could  be 
secured.  This  toxin  killed  rabbits  and  guinea-pigs  in  doses  varying 
from  0.5  to  i  cc. 

It  is  undoubtedly  an  endotoxin  that  is  liberated  from  the  bodies  of 
the  pneumococci  as  they  undergo  autolysis  or  are  dissolved  by  the 
enzymic  action  of  the  body  juices  or  the  cells.  The  toxin  liberated 
by  autolysis  has  been  carefully  studied  by  Rosenow,f  who  "  finds  it 
soluble  in  ether.  It  is  formed  during  retrogressive  changes  in  the 
pneumococci.  Heating  the  clear  autolysate  to  6o°C.  for  twenty 
minutes  destroys  it,  while  toxic  pneumococcus  suspensions  remain 
toxic  even  after  boiling.  Hydrochloric  acid  in  weak  solutions  de- 
stroys the  toxicity  of  pneumococcus  autolysates.  The  toxic  sub- 
stance is  absorbed  by  blood  charcoal  from  which  it  can  again  be 
obtained  by  shaking  with  ether.  Autolyzed  virulent  pneumococci 
and  non-virulent  pneumonia  diminish  the  toxicity  slightly  while 
unautolyzed  virulent  pneumococci  increase  it.  The  toxic  sub- 
stance is  probably  a  base  which  contains  amino  groups  of  nitrogen. 
Indications  have  been  obtained  showing  that  during  pneumococcus 
infections  toxic  substances  are  produced  that  do  not  call  forth  any 
immunizing  response."  Rosenowf  found  that  the  autolysate  con- 
tained a  proteolytic  enzyme.  He  also  found  §  that^it  was  capable 
of  producing,  in  dogs,  symptoms  strikingly  like  anaphylaxis,  with 
a  striking  drop  in  the  blood  pressure,  pronounced  hemorrhages, 
marked  depression  of  respiration,  extreme  cyanosis  and  the  pres- 
ence of  CO2  in  the  stomach.  .  ., 

Pathogenesis.  —  If  a  small  quantity  of  a  pure  culture  of  the  viru- 
lent organism  be  introduced  into  a  mouse,  rabbit,  or  guinea-pig,  the 
animal  dies  in  one  or  two  days.  Exactly  the  same  result  can  be  ob- 
tained by  the  introduction  of  a  piece  of  the  lung-tissue  from  croupous 
pneumonia,  by  the  introduction  of  some  of  the  rusty  sputum,  and 
frequently  by  the  introduction  of  human  saliva.  Postmortem  ex- 
amination of  infected  animals  shows  an  inflammatory  change  at  the 
point  of  subcutaneous  inoculation,  with  a  fibrinous  exudate  similar 
to  that  succeeding  subcutaneous  inoculation  with  the  diphtheria 
bacillus.  At  times,  and  especially  in  dogs,  a  little  pus  may  be  found. 
The  spleen  is  enlarged,  firm,  and  red-brown.  The  blood  with  which 
the  cavities  of  the  heart  are  filled  is  firmly  coagulated,  and,  like  that 
in  other  organs  of  the  body,  contains  large  numbers  of  the  bacteria, 

*  Ibid.,  1906,  ii. 

t"  Journal  of  Infectious  Diseases,"  1912,  x.  94,  235. 
j"  Journal  of  Infectious  Diseases,"  1912,  x.  287. 
§  Ibid.,  p.  480. 


450  Pneumonia 

most  of  which  exhibit  a  lanceolate  form  and  have  distinct  capsules. 
The  disease  is  thus  shown  to  be  a  bacteremia  unassociated  with 
conspicuous  tissue  changes. 

In  such  cases  the  lungs  show  no  consolidation.  Even  if  the  in- 
oculation be  made  by  a  hypodermic  needle  plunged  through  the 
breast-wall  into  the  pulmonary  tissue,  pneumonia  rarely  results. 
Gamaleia*  reported  that  pneumonic  consolidation  of  the  lungs  of 


Fig.  1 70. — Lung  of  a  child,  showing  the  appearance  of  the  organ  in  the  stage 
of  red  hepatization  of  croupous  pneumonia.  The  pneumonia  has  been  preceded 
by  chronic  pleuritis,  which  accounts  for  the  thickened  fibrous  trabeculae  extend- 
ing into  the  tissue,  and  which  may  have  had  something  to  do  with  the  peculiarly 
prominent  appearance  of  the  bronchioles  throughout  the  lung. 

dogs  and  sheep  could  be  brought  about  by  injecting  the  pneumococ- 
cus  through  the  chest- wall  into  the  lung.  Tchistowitschf  stated 
that  by  intratracheal  injections  of  cultures  into  dogs  he  succeeded  in 
producing  in  7  out  of  19  experiments  typical  pneumonic  lesions. 
MontiJ  claimed  to  have  found  that  a  characteristic  croupous  pneu- 

*  "Ann.  de  1'Inst.  Pasteur,"  1888,  n,  440. 

t  Ibid.,  1890,  in,  285. 

I  "Zeitschrift  fur  Hygiene,"  etc.,  1892,  xi,  387. 


Pathogenesis  451 

monia  results  from  the  injection  of  cultures  into  the  trachea  of  sus- 
ceptible animals.  A  very  interesting  review  of  the  literature  of  the 
experimental  aspects  of  the  subject,  embracing  198  references,  will 
be  found  in  Wadsworth's  paper  upon  "Experimental  Studies  on  the 
Etiology  of  Acute  Pneumonitis."* 

The  final  proof  that  true  pulmonary  consolidation,  i.e.,  pneumonia, 
can  be  produced  experimentally  by  cultures  of  the  pneumococcus  is 
to  be  found  in  a  paper  byLamarandMeltzer.f  These  investigators 
etherized  dogs,  kept  the  mouth  open  by  means  of  a  large 
wooden  gag,  drew  the  tongue  forward  by  means  of  hemostatic 
forceps,  and  then,  seizing  the  median  glosso-epiglottic  fold, 
pulled  it  forward  so  that  the  posterior  aspect  of  the  epi- 
glottis presented  an  inclined  plane.  Into  this  concavity  one  end 
of  a  tube  is  placed.  Under  the  protection  of  the  left  index- 
finger  the  tube  was  directed  into  the  larynx  and  pushed  down 
slowly  and  gently  through  the  trachea  until  a  resistance  was  met 
with.  The  inner  end  of  the  tube  was  then  found  to  engage  in  a 
bronchus — usually  the  right  bronchus.  A  pipette  containing  a  liquid 
culture  of  the  pneumococcus  was  next  attached  to  the  external  end 
of  the  tube,  and  by  means  of  a  syringe  the  culture  (about  6  cc.)  was 
injected  into  the  bronchus.  The  syringe  was  then  removed,  the 
piston  withdrawn,  and  the  syringe  again  attached  to  the  pipette.  By 
the  injection  of  air  the  culture  was  driven  deeper  into  the  bronchi. 
The  tube  was  then  clamped  and  withdrawn  and  the  animal  released. 
By  these  means  experimental  pneumonia,  with  the  typical  consolida- 
tion and  lobar  distribution,  was  produced  in  42  successive  cases.  The 
course  of  the  inflammatory  disturbance  thus  produced  was  rapid ,  and  in 
one  case  nearly  complete  consolidation  had  occurred  in  seven  hours. 

Lesions. — The  lesions  of  croupous  pneumonia  of  man  are  almost 
too  well  known  to  need  description.  The  distribution  of  the  disease 
conforms  more  or  less  perfectly  to  the  divisions  of  the  lung  into 
lobes,  one  or  more  lobes  being  affected.  An  entire  lung  may  be 
affected,  though,  as  a  rule,  the  apex  escapes  consolidation  and  is 
simply  congested.  The  invaded  portion  of  the  lung  is  supposed  to 
pass  through  a  succession  of  stages  clinically  described  as  (i)  con- 
gestion, (2)  red  hepatization,  (3)  gray  hepatization,  and  (4)  resolu- 
tion. In  the  first  stage  bloody  serum  is  poured  out  into  the  air-cells, 
filling  them  with  a  viscid  reddish  exudate.  In  the  second  stage  this 
coagulates  so  that  the  tissue  becomes  solid,  airless,  and  approxi- 
mately like  liver  tissue  in  appearance.  The  third  stage  is  charac- 
terized by  dissolution  of  the  ery  throcy  tes  and  invasion  of  the  diseased 
air-cells  by  leukocytes,  so  that  the  color  of  the  tissue  changes  from 
red  to  gray.  At  the  same  time  the  coagulated  exudate  begins  to 
soften  and  leave  the  air-cells  by  the  natural  passages,  and  the  stage 
of  resolution  begins. 

*  "Jour.   Amer.  Med.  Sciences,"  1904,  cxxvn,  p.  851. 
t  "Jour.  Exp.  Med.,"  1912,  xv,  No.  2,  p.  133. 


452  Pneumonia 

The  pneumococci,  though  present  in  enormous  numbers  in  the 
pulmonary  lesions,  are  not  confined  to  them.  In  practically  all 
cases  pneumonia  is  a  blood  infection  (bacteremia)  as  well  as  a  pul- 
monary infection.  It  is  through  the  blood  infection  that  many  of 
the  complications  and  sequelae  of  the  disease  are  brought  about. 

The  pneumococcus  is  not  infrequently  discovered  in  diseased  con- 
ditions other  than  croupous  pneumonia;  thus,  Foa,  Bordoni-Uffre- 
duzzi,  and  others  found  it  in  cerebro-spinal  meningitis;  Frankel,  in 
pleuritis;  Weichselbaum,  in  peritonitis;  Banti,  in  pericarditis;  numer- 
ous observers,  in  acute  abscesses;  Gabbi  isolated  it  from  a  case  of 
suppurative  tonsillitis;  Axenfeld  observed  an  epidemic  of  conjunc- 
tivitis caused  by  it;  Zaufal,  Levy,  and  Schroder  and  Netter  have 
been  able  to  demonstrate  it  in  the  pus  of  otitis  media,  and  Foulerton 
and  Bonney*  isolated  it  from  a  case  of  primary  infection  of  the 
puerperal  uterus.  It  has  also  been  found  in  arthritis  following  pneu- 
monia, and  in  primary  arthritis  without  previous  pneumonia  by 
Howard,  f 

Interesting  statistics  concerning  the  relative  frequency  of  pneumo- 
coccus infections  in  adults  given  by  Netter {  are  as  follows: 

Pneumonia 65.95 

Broncho-pneumonia 15  . 85 

Meningitis 13 .  oo 

Empyema 8 . 53 

Otitis  media 2  . 44 

Endocarditis 1.22 

Hepatic  abscess 1.22 

In  46  consecutive  pneumococcus  infections  of  children  he  found: 

Otitis  media 29 

Broncho-pneumonia 12 

Meningitis 2 

Pneumonia i 

Pleurisy i 

Pericarditis i 

Susceptibility. — Not  all  animals  are  equally  susceptible  to  the 
action  of  the  pneumococcus.  Mice  and  rabbits  are  highly  sensitive; 
dogs,  guinea-pigs,  cats,  and  rats  are  much  less  susceptible,  though  they 
may  also  succumb  to  the  inoculation  of  large  doses. 

Specificity. — The  etiologic  relationship  of  the  pneumococcus  to 
pneumonia  is  based  chiefly  upon  the  frequency  of  its  presence  in 
croupous  pneumonia.  Netter§  found  it  82  times  in  82  autopsies 
upon  such  cases;  Klemperer,  21  times  out  of  21  cases  studied  by 
puncturing  the  lung  with  a  hypodermic  syringe.  Weichselbaum  ob- 
tained it  in  94  out  of  129  cases;  Wolf,  in  66  out  of  70;  and  Pierce, 
in  no  out  of  121  cases.  In  about  5  per  cent,  of  the  cases  it  remains 
localized  in  the  respiratory  apparatus;  in  95  per  cent.,  it  invades  the 

*  "Trans.  Obstet.  Soc.  of  London,"  1903,  part  n,  p.  128. 
t  "Johns  Hopkins  Hospital  Bulletin,"  Nov.,  1903. 
t"  Compte-rendu,"  1889. 
§  "Compte-rendu,"  i88q. 


Specificity 


453 


blood.  An  interesting  paper  upon  this  subject  has  been  written  by 
E.  C.  Rosenow.* 

The  conditions  under  which  it  enters  the  lung  to  produce  pneu- 
monia are  not  known.  It  is  probable  that  some  systemic  depravity 
is  necessary  to  establish  susceptibility,  and  in  support  of  this  view 
we  may  point  out  that  pneumonia  is  very  frequent,  and  exceptionally 
severe  and  fatal,  among  drunkards,  and  that  it  is  the  most  frequent 
cause  of  death  among  the  aged.  Whether,  however,  any  particular 
form  of  vital  depression  is  necessary  to  predispose  to  the  disease, 
further  study  will  be  required  to  tell. 

Virulence. — Pneumococci  vary  greatly  in  virulence,  and  rapidly 
lose  this  quality  in  artificial  culture.  When  it  is  desired  to  maintain 
or  increase  the  virulence,  a  culture  must  be  frequently  passed  through 
animals.  Washbourn  found,  however,  that  a  pneumococcus  isolated 


Fig.  171. — Diplococcus    pneumoniae.     Colony    twenty-four    hours    old    upon 
gelatin.      X     100    (Frankel    and    Pfeiffer). 

from  pneumonic  sputum  and  passed  through  one  mouse  and  nine 
rabbits  developed  a  permanent  virulence  when  kept  on  agar-agar 
so  made  that  it  was  not  heated  beyond  ioo°C.,  and  alkalinized  4  cc. 
of  normal  caustic  soda  solution  to  each  liter  beyond  the  neutral  point 
determined  with  rosolic  acid.  The  agar-agar  is  first  streaked  with 
sterile  rabbit's  blood,  then  inoculated.  The  cultures  are  kept  at 
37-5°C.  Ordinarily  pneumococci  seem  unable  to  accommodate 
themselves  to  a  purely  saprophytic  life,  and  unless  continually  trans- 
planted to  new  media  die  in  a  week  or  two,  sometimes  sooner. 
Lambert  found,  however,  that  in  Marmorek's  mixture  (bouillon  2 
parts  and  ascitic  or  pleuritic  fluid  i  part)  the  organisms  would  some- 
times remain  alive  as  long  as  eight  months,  preserving  their  virulence 
during  the  entire  time. 

*  "Jour.  Infectious  Diseases,"  1904,  I,  p.  280. 


454  Pneumonia 

Virulence  can  also  be  retained  for  a  considerable  time  by  keeping 
the  organisms  in  the  blood  from  an  infected  rabbit,  hermetically 
sealed  in  a  glass  tube,  on  ice. 

Bacteriologic  Diagnosis. — It  is  usually  unnecessary  to  call  upon 
the  bacteriologist  to  assist  in  making  the  diagnosis  of  pneumonia. 
If,  for  any  reason  it  be  considered  necessary,  three  means  are 
available:  i,  the  blood  culture;  2,  the  inoculation  of  animals 
with  the  expectoration:  3,  the  cultivation  of  the  organism  from  the 
expectoration. 

1.  To  make  the  blood  culture,  the  elbow  is  encircled  with  a  band, 
the  skin  washed  and  after  an  application  of  iodine  has  been  made, 
a  hollow  needle  is  introduced  into  one  of  the  distended  veins,  and 
the  blood  permitted  to  drop  into  a  small  flask  or  tube  of  appropriate 
media. 

2.  To  inoculate  an  animal  with  the  sputum,  or  with  fluid  drawn 
from  the  lung  or  pleura.     A  white  mouse  or  a  rabbit  can  be  selected 
as  suitable.     Both  animals  are  so  susceptible  that  the  introduction  of 
one  drop  beneath  the  skin  is  usually  fatal  in  twenty-four  to  forty- 
eight  hours. 

Caution  must  be  exercised  in  using  this  means  of  diagnosis,  how- 
ever, as  the  pneumococcus  sometimes  occurs  in  normal  saliva, 
and  is  a  common  associated  organism  in  tuberculosis  and  other 
respiratory  diseases. 

3.  The  recovery  of  the  organism  from  the  sputum  can  be  accom- 
plished by  stroking  appropriate  media  with  a  platinum  wire  dipped 
in  the  sputum.     The  characteristic  colonies  can  be  picked  up   and 
transplanted  as  soon  as  they  appear. 

Identification  of  the  Organism. — Wads  worth*  has  been  able  to 
show  that  agglutination  reactions  can  be  obtained  by  concentrating 
the  pneumococci  in  isotonic  solution  and  adding  the  serum.  The 
method  does  not  seem  easily  applicable  for  diagnosis.  Neufeldf  and 
WadsworthJ  have  also  found  that  when  rabbit's  bile  is  added  to  a 
pneumococcus  culture  so  as  to  produce  lysis  of  the  organisms,  the  ad- 
dition of  pneumococcus-immune  serum  to  the  clear  fluid  so  obtained 
results  in  a  specific  precipitation.  This  seems  to  have  little  practical 
importance,  however,  for  purposes  of  diagnosis.  It  is,  however,  of 
some  importance  in  assisting  in  the  recognition  of  the  pneumococcus 
and  differentiating  it  from  the  streptococcus,  for  when  the  latter 
organisms  are  similarly  treated  no  precipitate  takes  place. 

Buerger§  found  that  all  pneumococci,  irrespective  of  source, 
were  agglutinated  by  pneumococcus-immune  serum,  that  such  serum 
was  capable  of  agglutinating  various  pyogenic  streptococci,  certain 
atypical  organisms,  and  certain  strains  of  Streptococcus  mucosus 
capsulatus.  The  sera  of  pneumonia  patients  varies  in  its  power  to 

*  "Jour.  Med.  Research,"  1904,  x,  p.  228. 

f'Zetschrift  fur  Hygiene,"  1902,  xi. 

%  Loc.  cit. 

§  "Jour.  Exp.  Med.,"  Aug.  25,  1905,  vn,  No.  5. 


Immune  Serum  455 

agglutinate  different  pneumococci;  some  strains  were  aggluti- 
nated, others  not.  The  sera  of  normal  individuals  and  of  normal 
rabbits  possess  no  agglutinating  power  for  pneumococci,  the 
atypical  organisms,  certain  streptococci,  or  Streptococcus  mucosus 
capsulatus. 

As  pneumococci  sometimes  grow  in  chains  instead  of  in  pairs,  and 
as  the  capsules  are  not  always  more  distinct  than  the  capsules  that 
sometimes  surround  streptococci,  it  may  be  necessary  to  resort  to 
special  methods  of  cultivation  for  the  final  identification  of  the  or- 
ganism. One  of  the  first  to  be  recommended  is  the  use  of  the  blood- 
agar  plate,  to  which  reference  has  been  made  in  the  section  upon 
Streptococcus  pyogenes. 

A  second  important  method,  and  one  that  not  only  differentiates 
the  pneumococcus  from  the  streptococcus,  but  from  the  common 
organisms  of  similar  morphology  that  infect  the  mouth,  is  the  inulin- 
serum  water  fermentation  test  of  Hiss.*  In  using  this  medium, 
Ruedigerf  found  it  best  prepared  as  follows:  Dissolve  5  gm.  of  NaCl, 
20  gm.  of  Witte's  peptone,  and  20  gm.  of  pure  inulin  in  1000  cc.  of 
distilled  water.  Add  20  cc.  of  a  5  per  cent,  solution  of  pure  litmus, 
and  tube,  putting  2  cc.  of  the  mixture  into  each  tube,  and  sterilize 
in  the  autoclave.  After  sterilization  add  (with  a  sterile  pipet)  2 
cc.  of  sterile,  heated  ascitic  fluid,  or,  preferably,  heated  beef-serum, 
to  each  tube,  and  incubate  twenty-four  hours  before  using.  Great 
care  must  be  taken  not  to  use  ascitic  fluid  that  contains  fermentable 
carbohydrates.  Each  lot  must  be  tested  with  some  strongly  fer- 
mentative bacterium,  and  the  absence  of  fermentable  carbohydrates 
proved.  Ruediger  prefers  this  preparation  to  the  original  solution 
of  Hiss  because  he  found  that  some  pneumococci  would  not  grow 
on  the  latter.  Fermentation  of  the  inulin  is  regarded  as  character- 
istic of  the  pneumococcus. 

The  pneumococcus  produces  red  colonies  upon  litmus-inulin-agar 
plates,  which  makes  their  use  desirable  when  pneumococci  are  to  be 
isolated  from  saliva,  throat  secretions,  or  other  material  in  which 
similar  appearing  organisms  are  apt  to  occur.  Ruediger  found  no 
other  mouth  bacteria  that  produced  red  colonies  on  these  plates. 

Immunity. — Pneumonia  is  peculiar  in  that  the  disease  in  human 
beings  terminates  by  crisis  as  though  from  some  source  a  supply  of 
antitoxin  or  other  immunizing  agent  was  suddenly  liberated,  but 
unfortunately  also  in  that  recovery  is  followed  by  immunity  of  such 
brief  duration  as  to  permit  the  occurrence  of  frequent  relapses.  It 
is  also  well  known  that  many  cases  show  a  subsequent  predisposition 
to  fresh  attacks  of  the  disease. 

Immune  Serum. — G.  and  F.  KlempererJ  have  shown  that  the 
serum  of  rabbits  immunized  against  the  pneumococcus  protects 

*  "Jour.  Amer.  Med.  Assoc.,"  1906,  vol.  XLVII,  p.  1171. 
t  "Jour,  of  Exp.  Med.,"  1905,  vol.  vi,  p.  317. 
.    J"  Berliner  klin.  Wochenschrift,."  1891,  Nos.  34  and  35. 


456  Pneumonia 

animals  infected  with  virulent  cultures.  When  applied  to  human 
medicine,  the  serum  failed  to  do  good. 

The  treatment  of  pneumonia  by  the  injection  of  blood-serum 
from  convalescent  patients,  tried  by  Hughes  and  Carter,*  has  been 
abandoned  as  useless  and  dangerous. 

Antipneumococcic  sera  have  been  experimentally  investigated  by 
De  Renzi,t  Washbourn,  J  and  Pane.§ 

Washbourn  prepared  an  antipneumococcus  serum  that  protected 
rabbits,  against  ten  times  the  fatal  dose  of  live  pneumococci, 
in  doses  of  0.3  cc.  In  general,  the  lines  upon  which  he  oper- 
ated were  those  of  Behring,  Marmorek's  work  with  the  streptococcus 
furnishing  most  of  the  details.  Two  cases  of  human  pneumonia 
seem  to  have  derived  some  benefit  from  large  doses  of  this  serum. 
The  sera  of  Pane  and  De  Renzi  were  not  so  powerful  as  those  of 
Washbourn,  requiring  about  i  cc.  to  protect  a  rabbit. 

McFarland  and  Lincoln||  succeeded  in  immunizing  a  horse  against 
large  doses  of  a  virulent  culture  of  the  pneumococcus,  and  obtained  a 
serum  of  which  0.5  to  0.25  cc.  protected  rabbits  from  many  times  the 
fatal  dose. 

The  experiments  by  Passler**  showed  some  gain  over  the  earlier 
work. 

The  antipneumococcic  sera  thus  far  produced  have  given  disap- 
pointing results  in  clinical  application. 

A  leukocytic  extract  prepared  by  Hiss  and  Zinsserff  from  an 
aleuronat  exudation  in  the  rabbit's  pleura  has  led  to  results  suf- 
ficiently encouraging  in  the  treatment  of  pneumonia  in  man  to  war- 
rant further  investigation  along  similar  lines. 

Rosenow|J  found  that  pneumococci  suspended  in  sodium  chlorid 
solutions  autolyse  rapidly.  By  means  of  this  autolysis  it  is  possible 
to  separate,  at  least  to  a  large  degree,  the  toxic  from  the  antigenic 
parts  of  the  pneumococcus,  as  the  toxic  part  goes  into  solution.  The 
injection  of  the  non-toxic  and,  as  it  appears,  antigenic  portion — auto- 
lyzed  pneumococci — causes  a  marked  increase  in  the  immunity  curve 
as  measured  by  the  specific  increase  in  pneumococcus  opsonin. 
The  injection  of  such  autolyzed  pneumococci  into  25  patients  with 
lobar  pneumonia  seemed  to  have  a  marked  beneficial  effect. 

Sanitation. — Pneumonia  is  undoubtedly  a  transmissible  disease. 
Exactly  how  infection  takes  place  is  not  known,  but  seeing  that  the 
infectious  agent  is  in  the  respiratory  tract,  from  which  it  is  easily 
discharged  into  the  atmosphere  during  cough,  etc.,  and  the  facility 

*  "Therapeutic  Gazette,"  Oct.  15,  1892. 
t  "II  Policlinic©,"  Oct.  31,  1896,  Supplement. 
:  "Brit.  Med.  Jour.,"  Feb.  27,  1897,  p.  510. 

§  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  May  29,1897,  xxi,  17  and  i8,p.  664. 
|]  "Jour.  Amer.  Med.  Assoc.,"  Dec.  16,  1899,  p.  1534. 

"Deutsches  Archiv  fur  klin.  Med.,"  Bd.  1905;  LXXXII,  Nos.  3,  4,  "Jour. 
Amer.  Med.  Assoc.,"  May  13,  1905,  p.  1538. 
("t  "Jour.  Med.  Research,"  1908,  xrx,  323. 
jt  "Jour.  Amer.  Med.  Assoc.,"  June  10,  LIV,  No.  24,  p.   1943. 


Bacillus  Capsulatus  Mucosus  457 

with  which  it  can  then  be  inhaled  by  those  nearby,  it  seems  justifiable 
to  conclude  that  the  primary  entrance  of  the  organism  into  the  body 
is  through  the  respiratory  tract.  Wood*  has  shown  that  "the  organ- 
isms in  the  sputum  do  not  remain  long  in  suspension  and  die  off 
rapidly  under  the  action  of  light  and  desiccation.  In  sunlight  or 
diffuse  daylight  the  bacteria  in  such  powder  die  within  an  hour,  and 
in  about  four  hours  if  kept  in  the  dark.  The  danger  of  infection 
from  powdered  sputum  may,  therefore,  be  avoided  by  ample  illu- 
mination and  ventilation  of  the  sick-room  in  order  to  destroy  or  dilute 
the  bacteria,  and  by  the  avoidance  of  dry  sweeping  or  dusting. 
Articles  which  may  be  contaminated  and  which  cannot  be  cleaned  by 
cloths  dampened  in  a  suitable  disinfectant  should  be  removed  from 
the  patient's  vicinity. 

PNEUMOCOCCUS    (FRIEDLANDER) — BACTERIUM    PNEUMONIA 
(ZopFf) — BACILLUS  CAPSULATUS  Mucosus  (FASCHING|) 

General  Characteristics. — An  encapsulated,  non-motile,  non-flagellated, 
non-sporogenous,  non-liquefying,  aerobic  and  optionally  anaerobic,  non-chromo- 
genic,  aerogenic  and  pathogenic  organism,  staining  by  ordinary  methods  but 
not  by  Gram's  method. 

This  organism  was  discovered  by  Friedlander§  in  1883  in  the 
pulmonary  exudate  from  a  case  of  croupous  pneumonia,  and,  being 
thought  by  its  discoverer  to  be  the  cause  of  that  disease,  was  called 
the  pneumococcus,  and  later  the  pneumobacillus.  The  grounds  upon 
which  the  specificity  of  the  organism  was  supposed  to  depend  were 
soon  found  to  be  insufficient,  and  the  organism  of  Friedlander  is  at 
present  looked  upon  as  one  whose  presence  in  the  lung  is,  in  most 
cases,  unimportant,  though  it  is  sometimes  associated  with  and  is 
probably  the  cause  of  a  special  form  of  pneumonia,  which,  ac- 
cording to  Stuhlern,||  is  clinically  atypical  and  commonly  fatal. 
Frankel  points  out  that  Friedlander's  error  in  supposing  his  organism 
to  be  the  chief  parasite  in  pneumonia  depended  upon  the  fact  that 
his  studies  were  made  by  the  plate  method,  which  permitted  the  dis- 
covery of  this  bacillus  to  be  made  more  easily  than  that  of  the  slowly 
growing  and  more  delicate  pneumococcus.  In  the  light  of  present 
knowledge  Friedlander's  bacillus  must  be  looked  upon  as  the  type 
of  a  group  of  organisms  varying  among  themselves  in  many  minor 
particulars. 

Distribution. — The  organism  is  sometimes  found  in  normal  saliva; 
it  is  a  common  parasite  of  the  respiratory  apparatus;  not  infrequently 
occurs  in  purulent  accumulations;  is  occasionally  found  in  feces,  and 
sometimes  occurs  under  external  saprophytic  conditions.  Thus  it  is 
probably  identical  with  the  "capsulated  canal- water  bacillus"  by 


'Jour.  Exp.  Med.,"  Aug.  25,  1905,  vn,  No.  5,  p.  624. 

'  Spaltpilze,"  1885,  p.  66. 

'Centralbl.  f.  Bakt.,"  1892,  etc.,  xn,  p.  304. 

'Fortshritte  der  Medizin,"  1883,  22,  715. 

'Centralbl.  f.  Bakt.,"  etc.  (Originale),  July  21,  1904,  Bd.xxxvi,  No.  4,  p.  493. 


45  8  Pneumonia 

Mori,*  and  may  belong  to  the  same  group  in  which  we  find  Bacillus 
aerogenes  capsulatus. 

.  Morphology. — Though  usually  distinctly  bacillary  in  form,  the 
organism  is  of  variable  length  and  when  paired  sometimes  bears  a 
close  resemblance  to  thepneumococcusof  Frankel  and  Weichselbaum. 
It  measures  0.5  to  1.5  IJL  in  breadth  and  0.6  to  0.5  /z  in  length.  It 
frequently  occurs  in  chains  of  four  or  more  elements  and  occasionally 
appears  elongated.  It  is  these  variations  in  form  that  have  led  to 
the  description  of  the  organism  by  different  writers  as  a  coccus,  a 
bacterium,  and  a  bacillus.  It  is  commonly  surrounded  by  a  distinct 
transparent  capsule,  hence  its  name  "capsule  bacillus"  and  Bacillus 
capsulatus  mucosus.  The  organism  is  non-motile,  has  no  spores, 
and  no  flagella.  It  stains  well  with  the  ordinary  anilin  dyes,  but 
does  not  retain  the  color  when  stained  by  Gram's  method. 


Fig.  172. — Bacterium    pneumonias    (modified    after    Migula). 

Cultivation. — Colonies. — If  pneumonic  exudate  be  mixed  with 
gelatin  and  poured  upon  plates,  small  white  spheric  colonies  appear 
at  the  end  of  twenty-four  hours,  and  spread  out  upon  the  surface  of 
the  gelatin  to  form  whitish  masses  of  a  considerable  size.  Under  the 
microscope  these  colonies  appear  irregular  in  outline  and  somewhat 
granular.  The  gelatin  is  not  liquefied. 

Bouillon. — There  is  nothing  characteristic  about  the  bouillon 
cultures  of  Friedlander's  bacillus.  The  medium  is  diffusely  clouded. 
A  pellicle  usually  forms  on  the  surface  and  a  viscid  sediment  soon 
accumulates. 

Gelatin  Puncture. — When  a  colony  is  transferred  to  a  gelatin 
puncture  culture,  a  luxuriant  growth  occurs.  Upon  the  surface  a 
somewhat  elevated,  rounded  white  mass  is  formed,  and  in  the  track 

*  " Zeitschrif t  fiir  Hygiene,"  1888,  rv,  p.  53. 


Bacillus  Capsulatus  Mucosus 


459 


of  the  wire  innumerable  little  colonies  spring  up  and  become  con- 
fluent, so  that  a  "  nail-growth  "  results.  No 
liquefaction  of  the  gelatin  occurs.  Gas  bub- 
bles not  infrequently  appear  in  the  wire 
track.  The  cultures  sometimes  become 
brown  in  color  when  old. 

Agar-agar. — Upon  the  surface  of  agar- 
agar  at  ordinary  temperatures  a  luxuriant 
white  or  brownish-yellow,  smeary,  viscid, 
circumscribed  growth  occurs. 

Blood-serum. — The  blood-serum  growth 
is  similar  to  that  upon  agar. 

Potato. — Upon  potato  the  growth  is  lux- 
uriant, quickly  covering  the  entire  surface 
with  a  thick  yellowish-white  layer,  which 
sometimes  contains  bubbles  of  gas. 

Milk  is  not  coagulated  as  a  rule.  Litmus 
milk  is  reddened. 

Vital  Resistance. — The  bacillus  grows  at 
a  temperature  as  low  as  i6°C.,  and,  accord- 
ing to  Sternberg,  has  a  thermal  death-point 
of  56°C. 

Metabolic  Products. — Friedlander's  ba- 
cillus ferments  nearly  all  the  sugars,  with 
the  evolution  of  much  gas.  It  generates 
alcohol,  acetic  and  other  acids,  and  both 
C(>2  and  H.  According  to  the  best  authori- 
ties the  organism  does  not  form  indol. 
There  is,  however,  some  difference  of  opin- 
ion upon  the  subject. 

Perkins*  divides  the  organisms  of  this 
group  into  three  chief  types  according  to 
their  reactions  toward  carbohydrates: 

I.  Bacillus  aerogenes  type  which  fer- 
ment all  carbohydrates,  with  the 
formation  of  gas. 

II.  Bacillus  pneumonias  (Friedlander) 
type  which  ferment  all  carbohy- 
drates except  lactose,  with  forma- 
tion of  gas. 

III.  Bacillus  lactis  aerogenes  type  which 
ferment  all  carbohydrates  except 
saccharose,  with  formation  of  gas. 
Pathogenesis. — Friedlander    found    con- 
siderable difficulty  in  producing  pathogenic 
changes  by  the  injection  of  his  bacillus  into  the  lower  animals. 


Fig.  173. — Friedlan- 
der's pneumobacillus; 
gelatin  stab  culture, 
showing  the  typical 
nail-head  appearance 
and  the  formation  of 
gas  bubbles,  not  always 
present  (Curtis). 


*  "Jour,  of  Infect.  Dis.,"  1904,  i,  No.  2,  p.  241. 


460  Pneumonia 

Rabbits  and  guinea-pigs  were  immune  to  its  action,  and  the  only 
important  pathogenic  effects  that  Friedlander  observed  occurred  in 
mice,  into  whose  lungs  and  pleura  he  injected  the  cultures,  with 
resulting  inflammation. 

That  Friedlander's  bacillus  may  be  the  cause  of  true  lobar  pneu- 
monia there  can  be  no  room  for  doubt  after  the  demonstrations  of 
Lamar  and  Meltzer,*  who  found  that  its  experimental  introduction 
into  the  bronchi  of  dogs  was  followed  by  true  lobar  pneumonia.  The 
lesions  in  these  dogs,  like  those  in  human  beings,  were  paler  in  color, 
the  lung  tissue  less  friable,  and  the  exudate  more  viscid  than  those 
caused  by  the  pneumococcus. 

Pneumonia  in  man,  caused  by  Bacillus  mucosus  capsulatus, 
is  atypical  clinically,  very  severe,  and  often  fatal. 

Curryf  found  Friedlander's  bacillus  in  association  with  the 
pneumococcus  in  acute  lobar  pneumonia;  in  association  with 
the  diphtheria  bacillus  in  otitis  media  associated  with  croup- 
ous  pneumonia;  and  in  the  throat  in  diphtheria.  In  pure  culture 
it  was  obtained  from  vegetations  upon  the  valves  of  the  heart  in 
a  case  of  acute  endocarditis  with  gangrene  of  the  lung;  from  the 
middle  ear,  in  a  case  of  fracture  of  the  skull  with  otitis  media;  and 
from  the  throat  in  a  case  of  tonsillitis.  Zinsser  has  twice  cultivated 
Friedlander's  bacillus  from  inflamed  tonsils  in  children. 

AbelJ  cultivated  it  from  the  discharges  of  fetid  ozena,  and  sup- 
posed it  to  be  the  specific  cause. 

Occasionally  Friedlander's  bacillus  bears  an  important  relation- 
ship to  lobular  or  catarrhal  pneumonia,  an  interesting  case  having 
been  studied  by  Smith.  §  The  histologic  changes  in  the  lung  were 
remarkable  in  that  the  "alveolar  spaces  of  the  consolidated  areas 
were  dilated  and  for  the  most  part  filled  with  the  capsule  bacilli." 
In  some  alveoli  there  seemed  to  be  pure  cultures  of  the  bacilli;  others 
contained  red  and  white  blood-corpuscles;  in  some  there  was  a  little 
fibrin.  The  bacillus  obtained  from  this  case,  when  injected  into  the 
peritoneal  cavity  of  guinea-pigs,  produced  death  in  eleven  hours. 
The  peritoneal  cavity  after  death  contained  a  large  amount  of  thick, 
slimy  fluid;  the  intestines  were  injected  and  showed  a  thin  fibrinous 
exudate  upon  the  surface;  the  spleen  was  enlarged  and  softened,  and 
the  adrenals  much  reddened.  Cover-glass  preparations  from  the 
heart,  blood,  spleen,  and  peritoneal  cavity  showed  large  numbers  of 
the  capsule  bacilli. 

Howard  1 1  has  also  called  attention  to  the  importance  of  this  bacil- 
lus in  connection  with  numerous  acute  and  chronic  infectious  proc- 
esses, among  which  may  be  mentioned  croupous  pneumonia,  suppura- 

*  "Jour.  Exp.  Med.,"  1912,  xv,  133. 

•  "Jour.  Boston  Soc.  of  Med.  Sci.,"  March,  1898,  vol.  n,  No.  8,  p.  137. 
j  "Zeitschrift  fur  Hygiene,"  xxi. 

§  "Jour.  Boston  Soc.  of  Med.  Sci.,"  May,  1898,  vol.  n,  No.  10,  p.  174. 
||  "Phila.  Med.  Jour.,"  Feb.  19,  1898,  vol.  i,  No.  8,  p.  336. 


1  Mixed  Pneumonias  461 

tion  of  the  antrum  of  Highmore  and  frontal  sinuses,  endometritis, 
perirenal   abscesses,   and  peritonitis. 

Virulence. — The  virulence  of  the  organism  seems  to  vary  under 
different  conditions.  It  is  sometimes  harmless  for  the  experiment 
animals,  but  when  injected  into  mice  and  guinea-pigs  usually  pro- 
duces local  inflammatory  lesions,  and  sometimes  death  from  septic 
invasion. 

CATARRHAL  PNEUMONIA  OR  BRONCHO -PNEUMONIA 

This  form  of  pulmonary  inflammation  occurs  in  local  areas,  commonly  situated 
about  the  distribution  of  a  bronchiole.  It  cannot  be  said  to  have  a  specific 
micro-organism,  as  almost  any  irritating  foreign  matter  accidentally  inhaled 
may  cause  it.  The  majority  of  the  cases,  however,  are  infectious  in  nature  and 
result  from  the  inspiration,  from  higher  parts  of  the  respiratory  apparatus,  of 
the  staphylococci  and  streptococci  of  suppuration,  Friedlander's  bacillus,  the 
bacillus  of  influenza,  and  other  well-known  organisms. 

TUBERCULOUS  PNEUMONIA 

The  progress  of  pulmonary  tuberculosis  is  at  times  so  rapid  that  the  tubercle 
bacilli  are  distributed  with  the  softened  infectious  matter  throughout  the  entire 
lung  or  to  large  parts  of  it,  and  a  distinct  pneumonic  inflammation  occurs.  Such 
a  pneumonia  may  be  caused  by  the  tubercle  bacillus,  or  the  tubercle  bacillus 
together  with  staphylococci,  streptococci,  tetragenococci,  pneumpcocci,  pneu- 
mobacilli,  and  other  organisms  accidentally  present  in  a  lung  in  which  ulceration 
and  cavity  formation  are  advanced. 

PLAGUE  PNEUMONIA 

The  pneumonic  form  of  plague  is  characterized  by  consolidation  of  the  lung 
histologically  and  anatomically,  indistinguishable  from  pneumococcic  and 
other  extensive  pulmonary  infections. 

MIXED  PNEUMONIAS 

It  frequently  happens  that  pneumonia  occurs  in  the  course  of  influenza  or 
shortly  after  convalescence  from  it.  In  these  cases  a  mixed  infection  by  the 
influenza  bacilli  and  pneumococci  is  commonly  found.  Sometimes  pneumococci 
and  staphylococci  simultaneously  affect  the  lung,  purulent  pneumonia  with 
abscess  formation  being  the  conspicuous  feature.  Almost  any  combination 
of  bacteria  may  occur  in  the  lungs,  so  that  it  must  be  left  for  the  student  to  work 
out  what  the  particular  effects  of  each  may  be. 

Among  the  mixed  forms  of  pneumonia  may  be  mentioned  those  called  by 
Klemperer  and  Levy  "complicating  pneumonias,"  occurring  in  the  course  of 
typhoid  fever,  etc. 


CHAPTER  XVII 
INFLUENZA 

BACILLUS  INFLUENZA  (R.  PFEIFFER) 

General  Characteristics. — A  minute,  non-motile,  non-flagellated,  non-sporpg- 
enous,  non-liquefying,  non-chromogenic,  aerobic,  pathogenic  bacillus,  staining 
by  the  ordinary  methods,  but  not  by  Gram's  method,  and  susceptible  of  artificial 
cultivation,  chiefly  through  the  addition  of  hemoglobin  to  the  culture-media. 

Notwithstanding  the  number  of  examinations  conducted  to 
determine  the  cause  of  influenza,  it  was  not  until  1892,  after  the  great 
epidemic,  that  Pfeiffer*  found,  in  the  blood  and  purulent  bronchial 
discharges,  a  bacillus  that  conformed,  in  large  part,  to  the  require- 
ments of  specificity. 

Morphology. — The  bacilli  are  very  small,  having  about  the  same 
diameter  as  the  bacillus  of  mouse  septicemia,  but  only  half  its  length 
(o.  2  by  o.  5  n) .  They  are  usually  solitary,  but  may  be  united  in  chains 
of  three  or  four. 

They  are  non-motile,  have  no  flagella,  and,  so  far  as  is  known,  do 
not  form  spores. 

Staining. — They  stain  rather  poorly  except  with  such  concentrated 
and  penetrating  stains  as  carbol-fuchsin  andLofHer's  alkaline  meth- 
ylene  blue,  and  even  with  these  more  deeply  at  the  ends  than  in  the 
middle,  so  that  they  appear  not  a  little  like  diplococci.  They  do  not 
stain  by  Gram's  method. 

Canon f  recommends  a  rather  complicated  method  for  the  demon- 
stration of  the  bacilli  in  the  blood.  The  blood  is  spread  upon  clean 
cover-glasses  in  the  usual  way,  thoroughly  dried,  and  then  fixed  by 
immersion  in  absolute  alcohol  for  five  minutes.  The  best  stain  is 
Czenzynke's: 

Concentrated  aqueous  solution  of  methylene  blue 40 

0.5  per  cent,  solution  of  eosin  in  70  per  cent,  alcohol 20 

Distilled  water 40 

The  cover-glasses  are  immersed  in  the  solution,  and  kept  in  the 
incubator  for  from  three  to  six  hours,  after  which  they  are  washed  in 
water,  dried,  and  mounted  in  Canada  balsam.  By  this  method  the 
erythrocytes  are  stained  red,  the  leukocytes  blue;  and  the  bacilli,  also 
blue,  appear  as  short  rods  or  as  dumb-bells. 

Large  numbers  of  bacilli  may  be  present,  though  sometimes  only  a 
few  can  be  found  after  prolonged  search,  as  they  are  prone  to  occur 

*  "Deutsche  med.  Wochenschrift,"  1892,  2;  "Zeitschrift  fur  Hygiene,"  1893, 
xiii,  357- 

f'Cehtralbl.  f.  Bakt.,"  etc.,  Bd.  xiv,  p.  860. 

462 


Cultivation 


463 


in  widely  scattered  but  dense  clusters.  They  are  frequently  inclosed 
within  the  leukocytes.  It  is  scarcely  necessary  to  pursue  so  tedious 
a  staining  method  for  demonstrating  the  bacilli,  for  they  stain  well 
enough  for  recognition  by  ordinary  methods. 

Isolation. — 'The  influenza  bacillus  grows  poorly  upon  artificial 
culture-media,  and  is  not  easy  to  isolate,  because  the  associated  bac- 
teria tend  to  outgrow  it.  When  isolated  it  is  difficult  to  keep,  as  it 
soon  dies  in  artificial  cultures. 

Pfeiffer  found  that  the  organism  grew  when  he  spread  pus  from 
the  bronchial  secretions  upon  serum-agar.  Subcultures  made  from 
the  original  colonies  did  not  "take."  By  a  series  of  experiments  he 
was  able  to  make  the  organism  grow  when  he  transferred  it  to  agar- 
agar,  the  surface  of  which  was  coated  with  a  film  of  blood  taken, 


Fig.  174. — Bacillus    of   influenza.     Smear   from    sputum  (after   Heim). 

with  precautions  as  to  sterilty,  from  the  finger-tip.  Later  it  was 
found  that  the  addition  of  hemoglobin  to  the  culture-medium  was 
equally  efficacious.  By  the  use  of  such  blood-smeared  agar  and 
glycerin-agar  the  organism  can  now  be  successfully  cultivated. 
The  isolation  is  best  achieved  through  the  use  of  bronchial  secre- 
tions, carefully  washed  in  sterile  water  or  salt  solution  to  remove 
contaminating  organisms  from  the  mouth. 

Cultivation. — Upon  blood-spread  glycerin  agar-agar,  after  twenty- 
four  hours  in  the  incubator,  minute  colorless,  transparent,  dewdrop- 
like  colonies  may  be  seen  along  the  line  of  inoculation.  They  look 
like  condensed  moisture,  and  Kitasato  makes  a  special  point  of  the 
fact  that  they  never  become  confluent.  The  colonies  may  at  times 
be  so  small  as  to  require  a  lens  for  their  detection. 

No  growth  takes  place  at  room  temperature.     The  organisms  die 


464  Influenza 

quickly  and  must  be  transplanted  every  three  or  four  days  if  they  are 
to  be  kept  alive. 

The  organism  is  aerobic  and  scarcely  grows  at  all  where  the  supply 
of  oxygen  is  not  free. 

In  bouillon  a  scant  development  occurs,  small  whitish  particles 
appearing  upon  the  surface,  subsequently  sinking  to  the  bottom  and 
causing  a ' '  wooly ' '  deposit  there.  The  bacillus  grows  more  luxuriantly 
upon  culture-media  containing  hemoglobin  or  blood,  and  can  be 
transferred  from  culture  to  culture  many  times  before  losing  vitality. 

Vital  Resistance. — Its  resisting  powers  are  very  restricted,  as  it 
speedily  succumbs  to  drying,  and  is  certainly  killed  by  an  exposure  to 
a  temperature  of  6o°C.  for  five  minutes.  It  will  not  grow  at  any 
temperature  below  28°C. 


Fig.  175. — Bacillus  of  influenza;  colonies  on  blood  agar-agar.     Low  magnifying 

power  (Pfeiffer). 

Specificity. — -From  the  fact  that  the  bacillus  is  found  chiefly  in 
cases  of  influenza,  that  it  is  present  as  long  as  the  purulent  secretions 
of  the  disease  last,  and  then  disappears,  and  that  Pfeiffer  was  able 
to  demonstrate  its  presence  in  all  cases  of  uncomplicated  influenza,  it 
seems  that  his  conclusion  that  the  bacillus  is  specific  is  justifiable. 
It  is  also  found  in  the  secondary  morbid  processes  following  influenza, 
such  as  pneumonia,  endocarditis,  middle-ear  disease,  meningitis,  etc. 
Horder*  has  cultivated  it  from  the  valvular  vegetations  of  2  cases  of 
endocarditis  following  influenza. 

Davisf  found  the  influenza  bacillus  in  the  respiratory  passage  of  a 
large  number  of  patients  suffering  from  whooping-cough. 

*  "Path.  Soc.  of  London,"  "Brit.  Med.  Jour.,"  April  22,  1905. 
f  "Jour.  Infectious  Diseases,"  1906,  in,  i. 


Immunity  465 

Pathogenesis. — The  bacillus  is  pathogenic  for  very  few  of  the 
laboratory  animals.  The  guinea-pig  is  susceptible  of  fatal  infec- 
tion, the  dose  required  to  cause  death  varying  considerably. 

Pfeiffer  and  Beck*  produced  what  may  have  been  influenza  in 
monkeys  by  rubbing  their  nasal  mucous  membranes  with  pure 
cultures. 

Immunity. — As  influenza  is  a  disease  that  commonly  relapses,  and 
from  which  one  rarely  seems  to  acquire  protection  against  future 
attacks,  there  must  be  scarcely  any  immunity  induced  through  ordi- 
nary infection.  Moreover,  the  organism  once  finding  its  way  into 
the  body  seems  to  remain  almost  indefinitely,  especially  when,  as  in 


Fig.  176. — Bacillus  of  influenza;  cover-glass  preparation  of  sputum  from  a  case 
of  influenza,  showing  the  bacilli  in  leukocytes.     Highly  magnified  (Pfeiffer). 

pulmonary  tuberculosis,  there  is  already  present  an  abnormal  con- 
dition furnishing  discharges  or  exudates  in  which  it  can  thrive. 

Delius  and  Kollef  found  that  the  toxicity  of  the  culture  does  not 
depend  upon  a  soluble  toxin,  but  upon  an  intracellular  toxin.  The 
outcome  of  the  researches,  which  were  made  most  painstakingly,  was 
total  failure  to  produce  experimental  immunity. 

Increasing  doses  of  the  cultures,  injected  into  the  peritoneal 
cavity,  enabled  the  animals  to  resist  more  than  a  fatal  dose,  but  never 
enabled  them  to  recover  when  large  doses  of  living  cultures  were 
administered. 

A.  Catanni,  Jr.,|  trephined  rabbits  and  injected  influenza  toxin 

*  "Deutsche  med.  Wochenschrift,"  1893,  xxi. 
t  "Zeitschrift  fur  Hygiene,"  etc.,  Bd.  1897,  xxrv,  Heft  2. 
j  Ibid.,  Bd.,  1896,  xxm. 
30 


466  Influenza 

into  their  brains,  at  the  same  time  trephining  control  animals,  into 
some  of  whose  brains  he  injected  water.  The  animals  receiving 
0.5  to  i  mg.  of  the  living  culture  died  in  twenty-four  hours  with  all 
the  nervous  symptoms  of  the  disease,  dyspnea,  paralysis  beginning 
in  the  posterior  extremities  and  extending  over  the  whole  body, 
clonic  convulsions,  stiffness  of  the  neck,  etc.  Control  animals  in- 
jected in  the  same  manner  with  water,  and  with  a  variety  of  other 
pathogenic  bacteria  never  manifested  similar  symptoms.  '  The  viru- 
lence of  the  bacillus  increased  rapidly  when  transplanted  from 
brain  to  brain. 

Diagnosis  of  Influenza. — Wynekoop*  employs  for  diagnosticating 
influenza  and  isolating  the  bacillus,  a  culture  outfit  similar  to  that 
used  for  diphtheria  diagnosis,  except  that  the  serum  contains  more 
hemoglobin.  The  swab  is  used  to  secure  secretions  from  the  pharynx 
and  tonsils,  and  from  the  bronchial  secretions  of  patients  with 
influenza,  then  rubbed  over  the  blood-serum.  In  many  such 
cultures  minute  colonies  corresponding  to  those  of  the  influenza 
bacillus  were  found.  Those  most  isolated  were  picked  up  with  a 
wire  and  transplanted  to  bouillon,  from  which  fresh  blood-serum 
was  inoculated  and  pure  cultures  secured. 

Carbol-fuchsin  was  found  most  useful  for  staining  the  bacilli. 
Wynekoop  observed  that  influenza  and  diphtheria  bacilli  sometimes 
coexist  in  the  throat,  and  that  influenza  bacilli  are  present  in  the  sore 
eyes  of  those  in  the  midst  of  household  epidemics  of  influenza. 

THE  PSEUDO-INFLUENZA  BACILLUS 

Pfeifferf  has  also  described  a  pseudo-influenza  bacillus — a  small,  non-motile, 
non-flagellated,  non-sporogenous,  Gram-negative  bacillus — that  he  found  in 
certain  cases  of  broncho-pneumonia  in  children.  It  differed  from  the  influenza 
bacillus  by  a  slightly  greater  size,  a  tendency  to  grow  in  chains,  and  to  undergo 
involution.  Martha  Wollsteint  believes  that  they  are  influenza  bacilli. 

*"  Bureau  and  Division  Reports,"  Department  of  Health,  city  of  Chicago, 
Jan.,  1899. 

t  "Zeitschrift  fur  Hygiene,"  etc.,  1892,  xm. 
t  "Jour.  Exp.  Med.,"  iqo6,  vni. 


CHAPTER  XVIII 
MALTA  OR  MEDITERRANEAN  FEVER 

MICROCOCCUS  MELITENSIS    (BRUCE);    BACILLUS    MELITENSIS 

(BABES) 

General  Characteristics. — A  non-motile,  non-flagellate,  non-sporogenous 
non-chromogenic,  non-liquefying,  pathogenic  coccus,  staining  by  the  ordinary 
methods,  but  not  by  Gram's  method;  characterized  by  remarkably  slow  growth 
and  by  pathogenic  action  upon  monkeys. 

In  1877,  while  working  in  Malta,  Bruce*  succeeded  in  finding  in 
every  fatal  case  of  Malta  fever  a  micrococcus  which  could  be  isolated 
in  pure  cultures  from  the  spleen,  liver,  and  kidney,  which  grew  readily 
on  artificial  media,  and  which,  when  injected  into  monkeys,  produced 
the  disease. 

Morphology. — 'Micrococcus  melitensis,  as  Bruce  called  it,  is  a 
round  or  slightly  oval  organism  measuring  about  0.3  p.  in  diameter. 
It  is  usually  single,  sometimes  in  pairs,  but  never  in  chains.  When 
viewed  in  the  hanging  drop  it  is  said  to  exhibit  active  "molecular" 
movements,  but  is  not  motile  and  has  no  flagella.  Babesf  declares 
it  to  be  a  bacillus. 

Staining. — It  stains  well  with  aqueous  solutions  of  the  anilin  dyes, 
but  not  by  Gram's  method. 

Thermal  Death  Point. — -This  has  been  fixed  by  Dal  ton  and  EyreJ 
at  57.S0C. 

Cultivation. — The  best  medium  for  its  cultivation  is  said  to  be 
ordinary  agar-agar.  After  inoculating,  by  a  puncture,  from  the 
spleen  of  a  fatal  case  of  Malta  fever,  the  tubes  should  be  kept  at  37°C. 
The  growth  first  appears  after  several  days,  in  the  form  of  minute 
pearly  white  spots  scattered  around  the  point  of  puncture  and  along 
the  needle  path.  After  some  weeks  the  colonies  grow  larger  and  join 
to  form  a  rosette-like  aggregation,  while  the  needle  tract  becomes 
a  solid  rod  of  yellowish-brown  color.  After  a  lapse  of  months  the 
growth  still  remains  restricted  to  the  same  area  and  its  color  deepens 
to  buff. 

When  the  sloping  surface  of  inoculated  agar-agar  is  examined  by 
transmitted  light,  the  appearance  of  the  colonies  is  somewhat  dif- 
ferent. At  the  end  of  nine  or  ten  days,  if  kept  at  37°C.,  some  of  the 
colonies  have  a  diameter  of  2  to  3  mm.  They  are  round  in  form,  have 
an  even  contour,  are  slightly  raised  above  the  surface  of  the  agar- 

*  "Practitioner,"  xxxiv,  p.  161. 

t  Kolle  and  Wassermann,  "Die  Pathogene  Mikroorganismen,"  in,  p.  443. 
t  "Jour,  of  Hygiene,"  1904,  iv,  p.  157. 

467 


468 


Malta  or  Mediterranean  Fever 


agar,  and  are  smooth  and  shining  in  appearance.  On  examining  the 
colonies  by  transmitted  light,  the  center  of  each  is  seen  to  be  yellow- 
ish, while  the  periphery  is  bluish-white  in  color.  The  same  colonies 
by  reflected  light  appear  milky-white.  Colonies  on  the  surface 
of  the  agar-agar  are  found  to  be  no  larger  than  hemp-seed  after 
a  couple  of  months  of  cultivation. 

When  kept  at  25°C.,  no  colonies  become  visible  to  the  naked  eye 
before  the  seventh  day;  at  37°C.,  before  the  third  or  fourth  day. 

In  bouillon  culture  kept  at  37°C.,  diffuse  clouding  of  the  medium 
occurs  in  three  or  four  days.  There  is  no  scum  on  the  surface.  No 
indol  is  formed.  In  sugar  bouillon  there  is  no  fermentation. 

In  milk  the  organism  grows  slowly  without  coagulation  and  with- 
out acid  production. 

The  growth  in  gelatin  takes  place  at  room   temperature  with 


•\ 


Fig.  177. — Micrococcus  melitensis. 

great  slowness,  first  appearing  in  about  a  month,  and  no  liquefac- 
tion of  the  medium  occurs. 

No  growth  takes  place  on  boiled  potato. 

Plate  cultures  are  not  adapted  to  the  study  of  the  organism  be- 
cause of  its  extreme  slowness  of  growth. 

Bacteriologic  Diagnosis. — The  specific  agglutinative  effect  of  the 
serum  can  be  made  use  of  for  the  purpose  of  diagnosis.  This  has 
been  studied  by  Wright,*  Birt  and  Lamb,f  and  later  by  Bassett- 
Smith.t 

All  of  the  observers  have  shown  that  the  agglutinative  reaction 
takes  place  both  with  living  and  dead  cultures  of  the  Micrococcus 
melitensis,  but  that  to  make  the  diagnosis  dilutions  of  serum  equal 
to  about  i  :  30,  never  greater  than  i  :  50,  must  be  used.  Birt  and 

*  "Lancet,"  1897,  March  6;  "Brit.  Med.  Jour.,"  1897,  May  15. 

t  Ibid.,  1899,  n,  p.  701. 

t  "British  Med.  Jour.,"  1902,  n,  p.  861. 


Treatment  469 

Lamb  also  arrive  at  certain  conclusions  regarding  the  prognosis 
based  upon  a  study  of  the  agglutinative  phenomena.  Their  conclu- 
sions are: 

1.  Prognosis  is  unfavorable  if  the  agglutinating  reaction  is  persistently  low. 

2.  Also  if  the  agglutinating  reaction  rapidly  fall  from  a  high  figure  to  almost 

zero. 

3.  A  persistently  high  and  rising  agglutinating  reaction  sustained  into  con- 

valescence is  favorable. 

4.  A  long  illness  may  be  anticipated  if  the  agglutination  figure,  at  first  high, 

decreases  considerably. 

The  agglutination  reaction  appears  early,  is  available  by  the 
end  of  the  first  week,  and  often  persists  for  years  after  convalescence. 

The  organisms  may  sometimes  be  cultivated  from  the  blood  taken 
from  a  vein,  but  are  more  certainly  to  be  secured  by  splenic  puncture. 

Pathogenesis. — The  micro-organism  is  not  pathogenic  for  mice, 
guinea-pigs,  or  rabbits,  but  is  fatal  to  monkeys,  goats,  dogs,  horses, 
asses,  and  mules,  when  agar-agar  cultures  are  injected  beneath  the 
skin. 

The  micro-organism  usually  seems  to  be  absent  from  the  circulat- 
ing blood,  though  Hughes  has  cultivated  it  from  the  heart's  blood 
of  a  dead  monkey. 

Bruce  not  only  succeeded  in  securing  the  micro-organism  from  the 
cadavers  of  Malta  fever,  but  has  also  obtained  it  during  life  by  splenic 
.puncture. 

Accidental  inoculation  with  Micrococcus  melitensis,  as  by  the 
prick  of  a  hypodermic  needle,  is  almost  invariably  followed  by  an 
attack  of  the  disease.  Six  cases  of  this  kind  in  human  beings  have 
occurred  in  connection  with  bacteriologic  work  on  Malta  fever  at 
Netley  and  two  additional  at  the  Royal  Naval  Hospital  at  Haslar 
and  in  the  Philippines.* 

Treatment. — The  treatment  of  Mediterranean  fever  by  means  of 
bacterio-vaccines  has  been  attempted  with  what  seems  to  be  glit- 
tering results  by  Bassett-Smith.f 

The  report  of  "British  Government  Commission  for  the  Investi- 
gation of  Mediterranean  Fever,"  published  by  the  Royal  Society, 
April,  1907,  has  greatly  elucidated  our  knowledge  of  the  pathogeny 
of  the  disease  by  showing  that  the  Micrococcus  melitensis  leaves  the 
body  of  the  patient  in  the  urine  and  in  the  milk.  It  has  not  been 
found  in  the  saliva,  sweat,  breath,  or  feces.  The  discovery  of  the 
organism  in  the  milk  suggested  that  it  might  be  through  milk  that  the 
specific  organisms  were  disseminated,  and  an  investigation  of  the 
goats  at  Malta,  where  the  disease  is  most  prevalent,  and  their  milk 
most  generally  used,  showed  that  a  large  percentage  of  the  animals 
were  infected  with  the  specific  cocci.  The  commission  has,  therefore, 
concluded  that  it  is  by  goats'  milk  that  the  disease  is  commonly 
disseminated,  though  they  point  out  that  fly-transmission  is  also 

*  See  Wright  and  Windsor,  "Jour,  of  Hygiene,"  1902,  n,  p.  413. 
t  "Journal  of  Hygiene,"  1907,  vn,  p.  115. 


470  Malta  or  Mediterranean  Fever 

possible.  In  the  Colonial  Office  Report  on  Mai  ta  in  1 907  it  was  shown 
that  over  40  per  cent,  of  the  goats  of  Malta  gave  the  serum  reaction, 
showing  that  they  had  had  the  disease,  while  10  per  cent,  of  them  were 
actually  secreting  the  cocci  in  their  milk.  The  authorities  permit  no 
milk  to  be  used  in  the  garrison  unless  it  is  boiled,  and  notice  that  by 
this  simple  measure  the  incidence  of  the  disease,  which  was  9.6  in 
1905,  had  fallen  to  2  in  the  corresponding  month  of  1906.  In  Report 
VII.  of  the  Mediterranean  Fever  Commission  (1906-07)  we  read: 

"The  epidemiologists  are  led  to  believe  that  quite  70  per  cent,  of  the  cases 
are  due  to  the  ingestion  of  goat's  milk."  In  their  opinion  ordinary  contact  with 
the  sick,  conveyance  of  infection  by  biting  insects,  house-flies,  dust,  drain  emana- 
tions, food  (other  than  milk),  and  water,  play  a  very  subordinate  part,  if  any, 
in  setting  up  Mediterranean  fever  in  man.  The  excellent  results  following  the 
preventive  measures  directed  against  goat's  milk  in  barracks  and  hospitals  also 
point  to  goat's  milk  as  being  the  chief  factor.  Among  the  soldiers  this  resulted 
in  a  diminution  of  about  90  per  cent. 

"For  example,  in  the  second  half  of  1905  there  were  363  cases  of  Mediter- 
ranean fever,  whereas  in  the  corresponding  part  of  1906  there  were  only  35  cases. 
Among  the  sailors  there  was  also  as  marked  a  fall  in  the  number  of  cases.  The 
Naval  Hospital  had  a  bad  reputation,  as  about  one- third  of  the  cases  of  fever 
occurring  in  the  fleet  at  Malta  could  be  traced  to  residence  in  this  hospital, 
either  as  patients  suffering  from  other  diseases  or  among  the  nursing  staff. 
The  goats  supplying  the  hospital  were  found  to  be  infected,  and  since  their  milk 
was  absolutely  forbidden,  not  a  single  case  of  Malta  fever  has  occurred  in  or 
been  traced  to  residence  in  this  hospital." 


CHAPTER  XIX 
MALARIA 

PLASMODIUM  MALARIA   (LAVERAN);  PLASMODIUM  VIVAX   (GRASSI 
AND  FELETTI)  ;  PLASMODIUM  FALCIPARUM  (WELCH) 

MALARIA,  or  paludism,  has  been  known  since  the  days  of  ancient 
medicine,  and  has  always  been  regarded  as  the  typical  miasmatic 
disease.  Its  name,  mala  aria,  means  "bad  air,"  and  is  Italian  de- 
rived from  the  Latin,  mains  and  aer,  coming  from  the  Greek  a^/o,  air, 
from  a«v,  to  blow.  The  other  name,  paludism,  from  the  Latin 
palus,  a  "marsh,"  refers  the  disease  to  the  bad  air  coming  from 
marshes. 

It  is  a  disease  of  extremely  wide  geographic  distribution,  and  since 
the  supposed  requirement,  marshy  ground,  is  found  in  nearly  all 
countries,  and  the  disease  is  particularly  prevalent  in  the  marshy 
districts  of  those  countries  in  which  it  occurs,  the  connection  between 
the  marshes  and  the  disease  seemed  clear.  Indeed,  the  two  are  inti- 
mately connected,  but  not  in  the  original  sense  as  will  be  shown  below. 

Both  hemispheres,  all  of  the  continents,  and  most  of  the  islands  of 
the  sea  surfer  more  or  less  from  malaria,  and  in  many  places, 
especially  in  the  tropics,  it  is  so  pestilential  as  to  make  the  country 
uninhabitable.  Probably  no  better  idea  of  the  wide  distribution 
and  severity  of  the  disease  can  be  obtained  than  by  reference 
to  Davidson's  "Geographical  Pathology."* 

The  disease  assumes  the  form  of  a  fever  of  intermittent  or  remittent 
type,  characterized  by  certain  peculiar  paroxysms.  When  typical, 
as  in  well-marked  intermittent  fever,  these  are  ushered  in  by  de- 
pression, headache,  and  chilly  sensations,  which  are  soon  followed  by 
pronounced  rigors  in  which  the  patient  shivers  violently,  his  teeth 
chattering.  The  temperature  soon  begins  to  rise  and  attains  a  height 
of  102°,  104°,  or  even  io6°F.,  according  to  the  severity  of  the  case. 
As  the  temperature  rises  the  sense  of  chilliness  disappears  and  gives 
place  to  burning  sensations.  The  skin  is  flushed,  hot,  and  dry. 
After  a  period  varying  in  length  the  skin  begins  to  break  out  into 
perspiration,  which  is  soon  profuse,  the  fever  and  headache  disappear 
and  the  patient  commonly  sinks  into  a  refreshing  sleep.  The 
frequency  of  the  paroxysms  varies  with  the  type  of  the  disease,  which, 
in  its  turn,  can  be  referred  to  the  kind  of  infection  by  which  it  is 
caused.  The  paroxysms  exhaust  the  patient  and  incapacitate  him 
and  may  eventually  prove  fatal,  though  in  by  far  the  greater  number 
of  cases  the  disease  gradually  expends  itself  and  a  partial  or  complete 
recovery  ensues.  Some  cases,  known  as  pernicious,  are  rapidly  fatal, 
*  D.  Appleton  &  Co.,  New  York,  1892. 


472  Malaria 

others  develop  into  a  chronic  cachexia,  with  profound  anemia  and 
complete  incapacitation  for  physical  or  mental  effort.  The  discovery 
of  Peruvian  or  Jesuits'  bark,  and  its  introduction  into  Europe  by  the 
Countess  del  Cinchon,  the  wife  of  the  Viceroy  of  Peru,  about  1639, 
marked  an  important  epoch  in  the  study  of  malarial  fever.  The 
isolation  of  its .  alkaloids,  quinin  and  cinchona,  begun  in  1810  by 
Gomez  and  perfected  in  1820  by  Pelletier  and  Coventou,  a  second 
great  epoch.  But  the  most  important  epoch  began  in  1880,  when 
Charles  Louis  Alphonse  Laveran,*  a  French  physician  engaged  in  the 
study  of  malarial  fever  in  Algeria,  announced  the  discovery  of  a 
parasite,  to  which  he  gave  the  name  Plasmodium  malarias,  in  the 
blood  of  patients  suffering  from  the  disease.  His  observations  were 
immediately  confirmed,  Biitschli  recognizing  the  parasitic  nature 
of  the  bodies  observed.  For  the  discovery  he  was  awarded  the  Bre- 
ant  prize. 

Laveran,  however,  threw  no  light  upon  the  source  of  infection,  and 
malaria  continued  to  be  described  as  a  miasmatic  disease. 

It  was,  however,  recognized  that  there  were  different  types  of  para- 
sites corresponding  to  the  different  clinical  forms  of  the  disease,  and 
Golgif  succeeded  in  correlating  the  various  appearances  of  the  para- 
sites so  as  to  express  their  life  cycles.  But  in  spite  of  the  interesting 
and  important  work  of  Golgi,  Celli,  Bignami  and  Marchiafava,  and 
many  others,  no  progress  was  made  in  accounting  for  the  entrance  of 
the  parasites  into  the  human  body. 

This  problem  had  long  interested  Sir  Patrick  Manson,  who  had 
devised  a  theory  which,  though  wrong  in  detail,  proved  in  the  end  to 
open  the  door  to  the  next  important  discovery.  Finding  that  the 
malarial  parasites  could  not  be  shown  to  leave  the  body  in  any  of  its 
eliminations,  and  remembering  that  the  same  was  true  of  the  filarial 
worms  and  their  embryos,  Manson  came  to  the  conclusion  that  they 
must  be  taken  out  of  the  blood  by  some  suctorial  insect.  The  one 
naturally  first  considered  was  the  mosquito,  which  was  known  to 
abound  wherever  malaria  prevailed.  Examining  mosquitoes  that 
had  been  permitted  to  distend  themselves  with  the  blood  containing 
the  parasites,  Manson  found  that  in  the  stomach  of  the  insect  the 
peculiar  phenomenon  known  as '"  flagellation,"  long  before  observed 
by  Laveran,  took  place  in  the  parasites,  giving  rise  to  long,  slender, 
lashing,  and,  finally,  free-swimming  filaments.  These,  he  conjec- 
tured, might  be  the  form  in  which  the  parasites  left  the  mosquito 
to  infect  the  swamp  water,  with  which  human  infection  eventually 
was  brought  about.  Here  Manson  failed,  but  while  he  was  investi- 
gating he  explained  the  whole  matter  to  Major  Ronald  Ross,  who 
was  soon  to  go  to  India,  and  whom  he  advised  to  make  the  matter  a 
subject  for  study  when  he  arrived  at  his  destination.  Rossf  ac- 
cepted the  opportunity  that  soon  presented  itself,  and,  after  a  most 

*  "  Accad.  d.  Med.,"  Paris,  Nov.  28  and  Dec.  28,  1880. 

t  "R.  Accad.  di  Medicina  di  Torino,"  1885,  xi,  20. 

j  "Indian  Medical  Gazette,"  xxxni,  14,  133,  401,  448. 


Malarial  Parasites  473 

painstaking  investigation,  the  details  of  which  are  given  in  a  paper 
which  can  be  found  in  the  International  Medical  Annual,*  1890, 
made  the  second  great  discovery  intheparasitologyof  malarial  fever. 
He  found  that,  as  Manson  thought,  the  mosquito  is  the  definitive 
host  of  the  parasite,  but  that  the  matter  is  much  less  simple  than  was 
imagined,  for  the  organisms  taken  up  by  the  mosquito  undergo  a 
complicated  life  cycle  requiring  about  a  fortnight  for  completion, 
after  which,  not  the  water  into  which  the  mosquito  might  fall  and  into 
which  its  contained  organisms  might  escape,  but  the  mosquito  it- 
self becomes  the  agent  of  infection.  In  other  words,  the  parasites 
taken  up  by  the  mosquito,  after  the  completion  of  the  necessary  de- 
velopmental cycle,  are  returned  by  the  mosquito  to  new.  human 
beings,  who  thus  become  infected.  Thus  it  was  shown  that  malaria 
is  not  a  miasmatic  disease  at  all,  but  that  it  is  an  infectious  disease 
whose  parasites  divide  their  life  cycle  between  man  and  the  mosquito, 
each  becoming  infected  by  the  other.  The  only  role  of  the  swamp  is 
to  furnish  the  mosquitoes,  and  since  these  are  only  more  numerous 
where  swamps  are  numerous,  but  may  occur  without  swamps,  the 
not  infrequent  occurrence  of  malarial  fevers  apart  from  swamps  is 
also  explained.  Ross  further  discovered  that  all  mosquitoes  are  not 
equally  susceptible  of  infection,  and,  therefore,  not  all  able  to  spread 
the  infection.  Indeed,  he  so  carefully  studied  the  mosquitoes  as  to 
narrow  the  infectability  and  infectivity  of  mosquitoes  down  to  one 
single  family,  the  Anophelinae,  and  to  one  single  genus,  Anopheles. 

There  remained,  however,  one  more  important  fact  to  be  eluci- 
dated, and  one  more  mysterious  body  to  be  accounted  for,  viz.,  the 
"flagellated"  body  that  had  misled  Manson.  This  was  found  by 
MacCallumf  to  be  but  the  spermatozoit  of  the  male  parasite.  While 
observing  one  of  the  malarial  parasites  of  birds — Plasmodium  dan- 
liewskyi — he  saw  one  of  these  "flagella"  swimming  away  from  its 
parent  parasite,  and  followed  it  carefully,  moving  the  slide  upon 
the  stage  of  the  microscope.  It,  and  others  of  its  kind,  approached  a 
large  globular  parasite,  to  which  one  effected  an  attachment  and  into 
which  it  entered.  MacCallum  realized  that  he  had  observed  the 
sexual  fertilization  of  the  organism.  In  1900  two  demonstrations 
of  momentous  importance  were  made.  First,  Sambon  and  Low 
went  to  Italy,  to  one  of  the  most  pestilential  parts  of  the  Campagna 
Romana,  and  lived  there  during  three  months  of  the  most  malarious 
4ime  of  the  year  in  a  mosquito-proof  house,  taking  every  precaution 
to  avoid  mosquitoes,  and  escaped  infection;  second,  anopheles 
mosquitoes  infected  in  Italy,  by  biting  malarial  patients,  were  taken 
to  England,  where  they  were  permitted  to  bite  Dr.  P.  J.  Manson 
and  Mr.  George  Warren,  both  of  whom,  after  a  period  of  incubation 
suffered  from  malarial  paroxysms  and  showed  plasmodia  in  their 
bloods.  What  may  perhaps  be  regarded  as  the  final  step  in  the  per- 

*  E.  B.  Treat  &  Co.,  New  York. 

t  "Journal  of  Exper.  Med.,"  1898,  m,  117. 


474 


Malaria 


faction  of  the  knowledge  of  the  parasite  was  reached  in  1911,  when 
C.  C.  Bass*  devised  a  method  of  cultivating  the  parasite  in  its 
asexual  stage,  in  vitro. 

Thus  from  its  time-honored  place  as  the  typical  miasmatic  disease, 
full  of  mystery  and  obscurity,  malarial  fever  suddenly  had  a  flood 
of  light  thrown  upon  it  by  which  every  peculiarity  was  fully 
illuminated. 

In  summarizing  the  knowledge  thus  set  forth  we  find  the  following 
facts: 

1880 — Discovery  of  the  Plasmodium  malariae  by  Laveran. 

1890 — Discovery  of  its  human  developmental  cycle  by  Golgi. 

1895 — Discovery  of  the  mosquito  cycle  and  mode  of  transmission 
by  Ross. 

1898 — Discovery  of  the  sexual  fertilization  of  the  parasite  by 
MacCallum. 

1911 — Discovery  of  the  method  of  cultivating  the  parasites  in 
vitro  by  C.  C.  Bass. 

The  interest  aroused  by  Laveran's  original  discovery  gave  a  great 
impetus  to  the  study  of  hematology  with  special  reference  to  para- 
sites, and  it  soon  became  evident  that  the  plasmodium  was  but  one 
of  a  group  of  similar  parasites.  Of  these  we  have  now  become  ac- 
quainted with  the  following: 


Parasite 

Plasmodium 
malarias. 


Plasmodium 
vivax. 


Plasmodium 
falciparum. 


Plasmodium 

kochi. 
Plasmodium 

inui. 
Plasmodium 

pitheci. 

Plasmodium 
brazilianum. 

Plasmodium 
cynomolgi. 

Plasmodium 
grassii         (Pro- 
teosoma  grassi). 

Plasmodium 
danliewskyi 
(Halteridium 
danliewskyi). 


Disease 
Quartan  fever. 


Tertian  fever. 


Aestivo-autumnal 
fever. 


Host 
Man. 


Man. 


Man. 


Cercopithicus. 


Insect  host 
Anopheles,     My- 
zorrhy  nchus, 
Mvzomyia,  Cel- 
lia" 

Anopheles,     My- 

zorrh  ynchus, 

Myzomyia, 

Cellia. 

Anopheles,       My- 

zorrhynchus, 
Myzomyia, 

Cellia. 
Unknown. 


Macacus      (Inuus     Unknown. 

cynomolgus). 
Orang     -     outang     Unknown. 

(Pithecus       sa- 

tyrus). 
Brachyrus  calores.     Unknown. 


Inuus  cynomolgus 

and  Inuus  nem- 

istrinus. 
Sparrows,    canary 

birds,  and  other 

small  birds. 
Owls,  hawks, 

crows,  and 

other          large 

birds. 


Unknown. 


Culex  pipens. 


Unknown. 


Journal  of  the  American  Medical  Association,"  1911,  XLVII,  1534. 


Malarial  Parasites 


475 


These  micro-organisms  correspond  in  all  essentials.  They  are 
protozoan  parasites  belonging  to  the  sporozoa  and  live  in  the  blood 
(hematozoa)  as  parasites  of  the  red  corpuscles.  They  all  have  two 
life  cycles,  one  which  is  asexual  in  the  intermediate  warm-blooded 
host,  and  one  that  is  sexual  in  the  definitive  cold-blooded  (insect) 
host.  Though  the  intermediate  hosts  vary  and  may  be  birds  or 


*i  ••'•'  SJ'^KSP 


Fig.  178. — Plasmodium  falciparum.     Ookinetes  in 

(Grassi). 


the  stomach  of  Anopheles 


mammals,  the  insect  hosts,  so  far  as  known,  are  always  mosquitoes. 
The  mosquitoes  become  infected  by  biting  and  sucking  the  blood  of 
infected  animals;  the  warm-blooded  animals  become  infected  by 
being  bitten  by  infected  mosquitoes,  and  so  on,  in  endless  cycles. 

The  parasites  differ  but  little  in  the  details  of  structure  and  de- 
velopment, so  that  the  following  description  may  serve  as  a  type 
for  all: 

From  the  proboscis  of  the  mosquito, 
with  its  saliva,  from  cells  in  the  salivary 
glands  where  they  have  been  harbored, 
tiny  elongate  spindles,  measuring  about 
1.5  M  in  length  and  0.2  ju  in  breadth,  and 
known  as  sporozoits,  enter  the  blood  of 
the  individual  bitten.  These  sporozoits 
attach  themselves  to  the  red  blood-cor- 
puscles, gradually  lose  their  elongate 
form,  and  become  irregularly  spherical. 
There  is  some  difference  of  opinion  as 
whether  the  little  bodies  are  simply 
upon  the  corpuscles,  as  Koch  believed, 
or  in  the  corpuscles,  as  the  majority 
of  writers  believe,  but  it  is  an  immate- 
rial difference,  for  the  parasite  soon 
makes  clear  that  it  is  consuming  the  corpuscle.  This  little  body 
is  known  as  a  schizont.  When  stained  with  polychrome  meth- 
ylene-blue,  and  examined  under  a  high  power  of  the  microscope,  it 
appears  as  a  little  ring  with  a  dark  chromatin  dot  upon  one  side. 
It  grows  steadily,  feeding  upon  the  hemoglobin,  which  seems  to  be 
chemically  transformed  into  fine  or  coarse  granules  of  a  bacillary  or 
rounded  form,  presumably  melanin.  In  a  length  of  time  that 


Fig.  179. — Plasmodium  fal- 
ciparum. Transverse  section 
of  the  stomach  of  Anopheles, 
showing  the  ookinetes  of  the 
parasite  in  various  stages  of 
development  attached  to  the 
outer  surface  (Grassi). 


22 


Fig.  180. — Developmental  cycle  of  plasmodium  vivax,  the  tertian  malarial 
parasite.  Figures  i  to  17  are  magnified  1200  diameters;  18  tot;,  only  600 
diameters:  i,  Sporozoit;  2,  penetration  of  a  sporozoit  into  a  red  UoocUcorpuscle; 
3  and  4,  schizont  developing  in  the  red  blood-corpuscles;  5  <lnd  6,  nuclear 
division  of  the  schizont;  7,  free  merozoits;  8  (following  the  arrows  to  the"  left  to 
3),  merozoits  entering  red  blood-corpuscles,  and  multiplying  by  schizOgony  3 
to  7;  after  longer  continuance  of  the  disease  the  sexual  forms  arise;  ga  to  i2a, 
macrogametocytes;  gbto  i2b,microgametocytes  still  in  the  circulatory  blood  of 
man.  If  the  macrogametocytes  (i2a)  are  not  taken  into  the  alimentary  canal  of 
the  mosquito,  they  multiply  parthenogenetically  (i2a,  I3C  to  i7c)  and  the 
resulting  merozoits  (i7c)  become  schizonts  (3  to  7).  The  figures  below  the 
dotted -line  represent  what  takes  place  in  the  alimentary  canal  of  anopheles 
(13  to  17);  i3b  and  i4b  the  formation  of  microgametocytes;  i3a  and  i3b,  matura- 
tion of  the  macrogametes;  isb,  a  microgamete:  16,  fertilization;  17,  ookinete; 


Malarial  Parasites  477 

varies — twenty-four  to  forty-eight  hours  (Plasmodium  falciparum), 
forty-eight  hours  (Plasmodium  vivax),  seventy- two  hours  (Plasmo- 
dium malarias) — the  schizonts  mature,  becoming  nearly  as  large  or 
quite  as  large  as  the  corpuscles.  The  pigment  granules  now  collect 
at  the  center  and  the  substance  of  the  parasite  divides  into  a  group 
of  equal-sized  merozoits,  commonly  known  as  spores.  Of  these 
there  are  usually  eight  in  the  meroblasts  of  Plasmodium  malariae, 
from  fifteen  to  twenty-five  in  those  of  Plasmodium  vivax,  and  from 
eight  to  twenty-five  in  Plasmodium  falciparum.  As  the  spores  be- 
come fully  formed  and  ready  to  separate,  the  paroxysm  of  the  disease 
begins.  It  ends  as  the  spores  are  freed  and  enter  new  corpuscles  to 
begin  the  cycle  over  again.  After  a  good  many  paroxysms  have 
occurred  it  may  be  observed  that  not  all  of  the  schizonts  change  to 
meroblasts  and  form  spores.  Some  remain  large  spheroidal  bodies  or, 
as  in  Plasmodium  falciparum,  assume  a  peculiar  crescentic  form  and 
remain  unchanged  in  the  blood.  These  are  the  sexual  parasites. 
The  female  is  usually  the  larger  and  is  known  as  the  makrogame- 
tocyte,  the  male,  the  smaller,  the  micro gametocyte.  These  are  the 
bodies  which,  when  removed  by  the  mosquito,  lay  the  foundation  of 
its  infection.  When  they  are  withdrawn  for  microscopic  examina- 
tion or  exposed  to  the  intestinal  juices  of  the  mosquito,  the  micro- 
gametocyte  becomes  tumultuous,  its  granules  are  observed  to  be  in 
a  state  of  active  cytoplasmic  streaming,  and  suddenly  there  burst 
forth  long  slender  filaments,  the  microgametes  or  spermatozoits. 
These  correspond  with  the  flagella  of  Laveran  and  others,  and  are 
the  same  bodies  that  Manson  thought  might  be  the  form  in  which 
the  parasite  leaves  the  insect's  body.  The  microgametes  lash  vig- 
orously for  a  time,  then,  breaking  loose,  swim  away,  and,  as 
MacCallum  observed,  conjugate  with  macro  gametes,  sexually  per- 
feet  cells  formed  from  the  macrogametocytes  by  "  reduction  divi- 
sion"  and  polar  body  formation,  thus  fertilizing  them.  As  there- 
suit  of  this  fertilization  a  zygote  or  ob'kinete  is  formed.  It  assumes  a 
somewhat  elongate  pointed  form  and  attaches  itself  to  the  wall  of 
the  mosquito's  stomach.  In  the  course  of  time  it  penetrates  and 
appears  upon  the  outside,  projecting  into  the  body  cavity.  It 
grows  larger  and  rounder,  divides  into  several  segments,  and  even- 
tually forms  an  ob'cyst  with  many  small  cells,  which  break  up  into 
myriad^^^iny  elongate  fusiform  bodies,  the  sporozoits.  These,  in 
the  course  of  i  ime,  seem  to  find  their  way  to  the  salivary  glands,  en- 
tering- into  Aie  epithelial  cells  and  taking  radial  positions  about  the 

18,  ookim^e^n  the  wall  of  the  mosquito's  stomach;  19,  penetration  of  the  gastric 
epithelium  by  the  ookinetes;  20  to  25,  stages  of  sporogenesis  on  the  outer  wall 
of  the  mosquito's  stomach;  2 6,  migration  of  the  sporozoits  to  the  salivary  glands 
of  the  mosquito;  27,  salivary  gland  with  sporozoits  in  the  epithelial  cells,  and 
escape  of  the  sporozoits  from  the  salivary  glands  through  the  insect's  proboscis 
at  the  time  a  human  host  is  bitten;  i,  free  sporozoit  from  the. mosquito's  saliva 
in  the  human  blood;  2,  penetration  of  the  sporozoit  into  a  red  blood-corpuscle, 
beginning  the  human  cycle  again  (Liihe). 

* 


478  Malaria 

nuclei,  where  they  remain  for  a  time.  Later,  they  leave  the  cells 
with  the  saliva,  and  when  the  mosquito  again  bites,  enter  the  warm- 
blooded host  to  infect  it,  if  of  the  appropriate  species. 

The  whole  cycle  in  the  mosquito  varies,  according  to  the  external 
temperature,  from  ten  days  to  a  fortnight.  The  mosquito  may  re- 
main alive  for  more  than  one  hundred  days,  and  must  bite  frequently 
to  satisfy  its  needs.  It  remains  infective  so  long  as  the  sporozoits 
remain  in  the  saliva,  which  is  usually  as  long  as  the  insect  is  alive. 
Here  it  may  be  remarked  that  as  it  is  only  the  female  mosquitoes 
that  bite,  it  is  only  by  them  that  the  infection  can  be  spread.  It  is 
an  interesting  question,  not  yet  solved,  whether  any  of  the  sporozoits 
entering  into  the  mosquito's  ovaries  can  infect  its  eggs  so  that  a  new 
generation  of  mosquitoes  may  be  born  infective. 

The  longer  the  human  infection  persists,  the  greater  the  number 
of  gametocytes  formed,  until  sometimes  in  aestivo-autumnal  malaria, 
no  schizonts  are  any  longer  found,  though  the  blood  contains  large 
numbers  of  gametocytes.  In  such  cases  the  gametocytes,  especially 
the  crescents  of  aestivo-autumnal  fever,  but  sometimes  also  those  of 
tertian  and  quartan  fever  undergo  regressive  schizogony,  by  partheno- 
genesis, in  the  patient's  blood,  and  without  fertilization  suddenly 
break  up  into  spores  which  enter  the  red  blood-corpuscles  and  occa- 
sion a  relapse  of  the  infection  that  had  apparently  spent  itself. 

THE  HUMAN  MALARIAL  PARASITES 

There  are  three  known  forms  of  human  malarial  parasites:  Plas- 

modium  malarias,  Plasmodium  vivax,  and  Plasmodium  falciparum. 

I.  Plasmodium  Malarias  (Laveran,*  1880). — This  is  the  smallest 

Synonyms. — Oscillaria  malariae  pro  parte,  Laveran,  1881.  Plasmodium  var. 
quartana,  Golgi,  1890.  Haemamceba  malariae.  Grassi  et  Feletti,  1892. 
Hasmamoeba  laverani  var.  quartana,  Labbe,  1894.  Plasmodium  malariae  quart- 
anum,  Labbe",  1899.  Haemomenas  malariae,  Ross,  1900.  Plasmodium  golgii, 
Sambon,  1902.  Plasmodium  quartanae,  Billet,  1904;  Celli,  1904.  ^ 

of  the  human  malarial  parasites.  Its  occurrence  is  relatively  infre- 
quent, as  is  that  of  the  quartan  fever  that  it  occasions.  The  schiz- 
ogonic  period  is  seventy-two  hours  long,  and  as  each  is  completed, 
a  paroxysm  of  the  disease  occurs. 

The  parasite,  in  the  red  blood-corpuscles,  first  appears  as 
ring,  at  one  side  of  which  there  is  a  chromatin  dot. 
organism  cannot  be  differentiated  from  Plasmodium 
end  of  twenty-four  hours  the  organism  seems  to  exten 
less  linearly,  and  sometimes  appears  as  a  long 
crosses  the  substance  of  the  unchanged  corpuscle. 
four  hours  the  breadth  of  the  parasite  is  two  or  three  times  as  great, 
and  it  has  become  pigmented.  The  corpuscle  itself  is  still  unchanged. 
In  the  last  twenty-four  hours  the  parasite  enlarges,  becomes  more  or 
less  quadrilateral,  finally  rounds  up,  shows  depressions  upon  the  sur- 

*  "Acad.  de  Med.,"  Nov.  23,  Dec.  28,  1880. 

* 


The  Human  Malarial  Parasites 


479 


face,  corresponding  to  the  divisions  into  which  it  is  to  segment,  the 
pigment  gathers  at  the  center,  and  the  substance  undergoes  cleavage 
resulting  in  the  formation  of  from  six  to  fourteen,  but  usually  eight, 
spores.  It  is  to  be  noticed  that  it  is  not  until  a  few  hours  before 
segmentation  that  the  parasite  becomes  as  large  as  the  corpuscle, 
and  that  the  corpuscle  is  never  enlarged  nor  bleached  by  the  presence 
of  the  parasite.  The  meroblasts  form  regular  rosettes,  or  "daisy- 
heads,"  within  the  corpuscles. 

In  single  infections  the  parasites  are  all  of  the  same  age  and  all 
mature  at  the  same  time,  so  that  in  any  examination  of  the  blood 
they  will  all  appear  uniform.  It  is,  however,  sometimes  true  that 
the  patient  may  have  been  infected  one  day  by  one  mosquito  bite, 
and  again  infected  the  next  day  or  the  third  day  by  a  second  mos- 
quito bite,  so  that  his  blood  contains  two  crops -of  the  microparasites, 
arriving  at  maturity  at  different  times.  This  perplexes  the  clinician 


Fig.  181.  —  Parasite  of  quartan  malarial  fever:  a,  b,  c,  d,  enlarging  intracellular 
parasites;  e,  /,  g,  h,  segmentating  parasites  forming  a  distinct  rosette  from  which 
the  spores  separate;  i,  macrogametocyte;  j,  microgametocyte;  k,  sporozoit. 

through  ihe  variety  of  parasitic  forms  in  the  blood  and  the  abnormal 
frequency  of  the  paroxysms. 

The  gametocytes  of  the  parasite  remain  for  some  time  in  the  red 
corpuscles  without  division,  but,  finally,  become  free  spherical  bodies. 
Two  sizes  can  be  made  out,  the  larger,  the  macrogametocyte  or 
female,  the  other,  the  microgametocyte  or  male.  Each  has  proto- 
with  a  tendency  to  take  a  blue-gray  color  and  appear  uni- 
granular,  except  that  at  some  part  of  the  periphery  of  each 
there  isj^reular  or  semicircular  area  that  is  free  from  granules. 
This  ^1  «irger  in  the  microgametocyte. 

II.Ba  rnMium  Vivax  (Grassi  and  Feletti,*  1890).—  -This  is  the 


malariae  pro  parte,  Laveran,  1881.  Plasmodium  var. 
tertiana/^oTgi,  1889.  Haemamceba  vivax,  Grassi  et  Feletti,  1890.  Haemamceba 
laverani  var.  tertiana,  Labbe,  1894.  Plasmodium  malarias  tertianum,  Labbe", 
1899.  Haemamceba  malariae  var.  magna,  Laveran,  1900.  Haemamceba  malariae 
var.  tertianae.  Laveran,  1904.  Plasmodium  tertianae  pro  parte,  Billet,  1904. 

most  common  of  the  malarial  parasites  of  man,  and  occasions  the 

"  benign  "  tertian  fever.     It  is  a  large  parasite,  the  full-grown  schizont 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1890,  vn,  396;  1891,  x,  449,  481,  517. 


480  Malaria 

(meroblast),  ready  to  form  merozoits,  and  the  gametocytes  all 
exceeding  the  size  of  the  red  blood-corpuscles.  It  matures  in  forty- 
eight  hours,  but  not  with  mathematic  precision.  In  single  infections 
the  greater  number  of  the  parasites  are  of  the  same  age  and  present 
the  same  appearance,  but  various  shapes  and  ages  may  be  found 
together.  In  double  infections,  with  paroxysms  every  day,  para- 
sites of  different  ages  may  be  found. 

The  youngest  form  in  which  the  parasite  can  be  observed  is  that 
of  a  tiny  ring  in  a  red  blood-corpuscle.  The  periphery  of  this 
ring  (when  the  blood  is  stained  with  polychrome  methylene  blue) 
is  outlined  with  blue,  at  one  side  there  is  a  distinct  blue  dot,  and 
the  center  appears  colorless  and  like  a  vacuole.  The  dot  is  usually 
on  the  side  of  the  vacuole  that  has  the  thinner  protoplasmic  outline. 
The  smallest  such  rings  usually  have  a  diameter  equal  to  about  H 
the  diameter  of  the  blood-corpuscle.  The  tiny  ring-form,  or,  as 
it  might  better  be  called,  the  "  seal-ring  form,"  continues  until  the 


66 


Fig.  182.  Fig.  183. 

Figs.  182, 183. — Gametocytes  of  plasmodium  malariae :  85,  The  macrogametocyte; 
86,  the  microgametocyte  (Kolle  and  Wassermann). 

schizont  becomes  half  the  diameter  of  the  blood-corpuscle,  when 
its  protoplasm  has  begun  to  increase  so  rapidly  that  the  vacuole 
no  longer  appears  to  be  so  conspicuous.  The  organism  also  becomes 
irregular  in  shape  and  is  actively  ameboid,  its  protoplasm  streaming 
this  way  and  that  when  examined  in  fresh  blood.  At  this  time  it 
may  be  noticed  that  the  infected  blood-corpuscle  is  increasing  in 
volume,  sometimes  becoming  twice  the  normal  size,  and  also  be- 
coming pale  in  color.  It  seems  also  as  though  the  disk  shape  of 
the  corpuscle  was  lost,  and  it  had  become  swollen  into  a  more 
spherical — sometimes  irregular — form.  The  parasite,  which  may 
still  show  a  relic  of  its  original  ring-form,  now  shows  plentifully 
throughout  its  protoplasm  exceedingly  fine  granules  of  yellow- 
brown  pigment.  When  from  thirty-six  to  forty  hours  old,  all  trace 
of  the  "seal-ring"  form  disappears,  the  ameboid  action  becomes  less 
marked,  and  the  parasites  (now  three-quarters  the  size  of  the  en- 
larged pale  and  misshapen  corpuscles  in  which  they  are  contained) 


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DESCRIPTION  OF  PLATES  II  AND  III. 


Various  forms  of  malarial  parasites:  Figs,  i  to  10  inclusive,  tertian 
parasites;  Figs,  n  to  19  inclusive,  quartan  parasites;  Figs.  20  to  26 
inclusive,  estivo-autumnal  parasites. 

i. — Normal  red  blood-cell.  2. — Young  tertian  ring.  3. — Large  ter- 
tian ring.  4. — Half -grown  tertian  parasite.  5. — Infected  cell  showing 
Schiiffner's  dots.  6. — Adult  tertian  parasite.  7. — Beginning  sporula- 
tion.  8. — Sporulation  completed.  9. — Tertian  microgametocyte.  10. 
— Tertian  macrogamete.  n. — Young  quartan  ring.  12. — Older  quar- 
tan ring  13.— Quartan  band.  14.— Older  quartan  band.  15. — Full- 
grown  quartan  parasite.  16. — Mature  parasite  with  divided  chromatin. 
17. — Sporulation  completed.  18. — Quartan  microgametocyte.  19.— 
Quartan  macrocyte.  20. — Young  estivo-autumnal  ring.  21. — Large 
estivo-autumnal  ring.  22. — Mature  parasite.  23. — Sporulation  com- 
pleted. 24. — Estivo-autumnal  microgametocyte.  25. — Estivo-autum- 
nal macrogamete.  26. — Estivo-autumnal  ovoid. 

(From  Deaderick,  "A  Practical  Study  of  Malaria.") 


PLATE  II 


o 


w  Vjl 

& 

4 


'  V\ 

fill" 


,» 
£« 


--•v  m 

9 


10 


12 


PLATE   III 


13 


14 


15 


•- 

^.«.^! 


17 


18 


19 


o 


O 


20 


21 


22 


23 


24. 


25 


26 


The  Human  Malarial  Parasites 


481 


appear  as  irregular,  ragged,  protoplasmic  bodies  filled  with  fine 
pigment  granules.  In  about  forty-five  hours  they  completely  fill 
the  enlarged  corpuscles,  and  begin  to  gather  their  protoplasm  into 
rounded  formations  in  which  the  pigment  is  no  longer  distributed, 
but  occurs  in  irregular  stripes  or  gathers  together  into  a  rounded 
clump.  In  a  couple  of  hours  the  blood-corpuscle  has  disappeared 
and  the  rounded  parasite,  larger  than  normal  red  corpuscles,  with 
a  lobulated  surface,  and  with  its  pigment  granules  collected  to 
form  one  or  two  rounded  masses,  is  seen  to  have  reached  the  stage 
of  the  meroblast.  This  does  not  form  the  rosette  or  " daisy-head" 
shown  by  the  quartan  parasite,  but.  might  better  be  compared  to  a 
mulberry,  and  eventuates  in  the  formation  of  from  fifteen  to  twenty- 
five  small,  rounded  or  ovoid,  pale,  unpigmented  bodies,  the  mero- 


Fig.  184. — Parasite  of  tertian  malarial  fever:  a,  b,  c,  d,  e,  f,  g,  Growing' pig- 
mented  parasite  in  the  red  blood-corpuscles;  h,  spores  formed  by  segmentation 
of  the  parasite — no  rosette  is  formed,  but  concentric  rings  of  the  cytoplasm  divide; 
*,  macrogametocyte;  i,  microgametocyte  with  spermatozoits. 

zoits  or  spores.  These  become  freed  from  the  pigment  and  at- 
tached to  new  red  corpuscles,  in  which  they  are  easily  recognized 
as  the  "tiny-rings"  that  begin  the  schizogonic  cycle.  The  game- 
tocytes  of  the  tertian  parasite,  the  "free  spheres,"  as  they  are  some- 
times called,  are  large,  rounded  or  slightly  ovoid  bodies,  with  a 
uniformly  dull  bluish-gray  or  grayish-green  protoplasm,  in  the  in- 
terior of  which  there  is  always  a  circular  or  semicircular  area  periph- 
erally or  centrally  situated,  and  colorless.  Except  in  this  area 
the  pigment  is  distributed  throughout  the  parasite.  The  larger 
or  macrogametocyte,  the  female  parasite,  measures  10  to  14  yu  in 
diameter.  It  has  a  greenish  or  grayish-green  or  almost  colorless 
protoplasm,  containing  an  oval  or  bean-shaped  colorless  area  al- 
most half  as  large  as  the  organism  itself.  Yellowish-brown  pig- 


482  Malaria 

ment  in  short,  broad  rods  is  sparingly  scattered  throughout  the 
substance  elsewhere. 

The  microgametocyte  or  male  form  is  approximately  the  size 
of  a  red  blood-corpuscle — 8  to  9  /JL  in  diameter.  It  stains  more  deeply 
than  its  mate  and  contains  more  and  coarser  pigment  granules. 

III.  Plasmodium    Falciparum     (Welch,*     1897). — This    is    the 

Synonyms. — Oscillaria  malariae  pro  parte,  Laveran,  1881.  Hsemamoeba  praecox, 
Grassi  et  Feletti,  1 890.  Laverania  malarias,  Grass!  et  Feletti,  1 890.  Haemamoeba 
malariae  praecox,  Grassi  et  Feletti,  1892.  Haemomenas  praecox,  Ross,  1899. 
Plasmodium  malariae  praecox,  Labbe,  1899.  Plasmodium  preecox,  R.  Blanchard, 
1900.  Haemamceba  malariae  var.  parva,  Laveran,  1900.  Plasmodium  immacu- 
latum,  Schaudinn,  1902.  Laverania  praecox,  Nocard  et  Leclainche,  1903. 

parasite  of  estivo-autumnal  or  malignant  tertian  malarial  fever. 
It  is  a  very  small  parasite,  whose  occurrence,  even  multiple  occur- 
rence, in  the  corpuscles  does  not  change  their  size  or  shape.  It 
does,  however,  quickly  change  the  appearance  of  the  corpuscles, 
which  become  polychromatophilic,  and  frequently  show  numerous 
small  dots — the  granulations  of  Schiiffner — in  the  corpuscular 
substance. 

The  first  appearance  of  the  schizont  is  in  the  form  of  tiny  rings, 
which  appear  to  lie  upon  rather  than  in  the  corpuscles,  and  are  first 
seen  at  the  edges.  The  rings  are  outlined  by  extremely  fine  lines 


Fig.  185.  Fig.  186. 

Figs.  185,  186. — Gametocytes  of  plasmodium  vivax:  87,  The  microgametocyte; 
88,  the  macrogametocyte  (Kolle  and  Wassermann). 

and  sometimes  seem  to  be  incompletely  closed,  so  that  they  are 
like  horseshoes  rather  than  circles.  They  increase  to  several  times 
the  original  size  without  losing  the  ring  shape,  and  are  variously 
known  as  "middle-sized  rings"  and  " large  rings."  They  are  with 
difficulty  differentiated  from  the  "tiny  rings"  of  the  tertian  parasite. 
As  the  "large  ring"  stage  is  reached  the  parasites  begin  to  disappear 
from  the  peripheral  blood  to  complete  their  growth  and  undergo 
meroblast  formation  in  the  capillaries  of  the  spleen,  the  brain,  and 
the  bone-marrow.  Here  the  full-grown  parasites — meroblasts — 
appear  as  irregular  disks,  resembling  those  of  the  quartan  parasite, 

*  Article  " Malaria "    in    "A    System   of    Practical  Medicine  by    American 
Authors,"  1897,  p.  138. 


The  Human  Malarial  Parasites 


483 


but  smaller  in  size.  The  pigment  is  gathered  toward  the  center 
in  a  little  mass,  and  eight  to  twenty-five  merozoits  are  formed  in  a 
morula  or  mulberry-like  mass  similar  to  those  of  the  tertian  para- 
site. Two  or  three  parasites  to  the  corpuscle  are  frequent.  They 
are  actively  ameboid,  do  not  mature  simultaneously,  and  hence 


Fig.  187. — Parasite  of  estivo-autumnal  fever:  a,  b,  c,  Ring-like  and  cross- 
like  hyaline  forms;  d,  e,  pigmented  forms;  /,  g,  segmentary  forms;  A,  i,  j, 
crescents. 

there  are  no  regularly  occurring  paroxysms.     The  duration  of  the 
asexual  cycle  is  from  twenty-four  to  forty-eight  hours. 

The  gametocytes  are  striking  and  characteristic  ovoid  and  cres- 
centic  bodies — crescents — i%  times  the  diameter  of  a  red  blood- 
corpuscle  in  length,  and  about  half  the  diameter  of  the  corpuscle 
in  breadth.  The  ends  color  more  intensely  with  methylene  blue 


Fig.  1 88.  Fig.  189. 

Figs.  188,  189. — Gametocytes   of  plasmodium   falciparum:   91,  The  microga- 
metocyte;  92,  the  macrogametocyte  (Kolle  and  Wassermann). 

than  the  middle  portion,  and  the  bacillary  pigment  granules  are 
collected  toward  the  centers.  The  longer  and  more  slender  crescents 
are  usually  bent,  and  the  relic  of  the  corpuscle  in  which  they  have 
formed  can  often  be  seen  forming  a  line  connecting  the  ends  on  the 
concave  side.  These  are  the  microgametocytes  or  male  elements. 


484  Malaria 

The  macrogametocytes  are  broader,  not  curved,  and  sometimes 
are  ovoidal  or  prolate  spheroidal  in  shape.  The  pigment  granules 
are  more  widely  scattered  throughout  the  substance.  The  cres- 
cents are  most  numerous  after  the  fever  has  lasted  for  some  time  or 
in  recurrences  of  the  fever. 

The  fever  in  this  form  of  malarial  infection  may  be  intermittent 
with  daily — 'quotidian — paroxysms,  or  with  irregular  paroxysms,  or 
the  fever  may  be  remittent.  The  infection  is  sometimes  mild,  but 
may  be  so  severe  as  to  be  rapidly  fatal.  In  such  cases  the  number 
of  parasites  is  enormous,  the  cerebral  capillaries  become  filled  with 
them,  and  coma  quickly  comes  on  and  is  soon  followed  by  death. 
Such  cases  are  described  as  " congestive  chills"  or  " algid"  cases. 

Cultivation  of  the  Parasites. — -The  parasites  have  been  successfully 
cultivated  in  blood,  prevented  from  coagulation,  by  Bass. 

In  the  first  paper,  Bass  announced  that  the  cultivation  of  these 
parasites  was  made  possible  by  the  maintenance  of  the  culture  at 
4o°C.,  the  selection  of  such  an  elevated  temperature  being  based 
upon  the  theory  that  in  the  bloods  of  infected  human  beings,  there 
were  specific  amboceptors  directed  against  the  invading  organisms, 
but  unable  to  effect  their  destruction  until  complement  is  formed. 
Complement  soon  appears  in  the  drawn  blood,  according  to  Bass, 
unless  the  temperature  be  sufficiently  elevated  to  prevent  it,  and  he 
finds  4o°C.  sufficient  for  the  purpose.  A  later  paper  by  Bass  and 
Johnsj  gives  the  details  of  cultivation  as  follows: 

When  blood  is  to  be  taken  from  a  malarial  patient  for  the  purpose  of  cultivat- 
ing the  parasites,  one  prepares  a  sterile  50  per  cent,  solution  of  Merck's  dex- 
trose, in  distilled  water,  and  measured  into  a  sterilized  test- tube,  i  inch  in  diameter 
o.i  cc.  for  each  10  cc.  of  blood  to  be  collected.  The  tube,  which  is  called  the 
" defibrinating  tube"  is  provided  with  a  glass  rod  that  passes  through  the 
cotton  plug  to  the  bottom  of  the  tube.  A  needle  is  plunged  into  the  arm  vein 
of  the  patient,  and  the  infected  blood  is  permitted  to  flow  into  the  defibrinating 
tube  until  the  requisite  quantity  has  been  collected.  The  needle  is  then  with- 
drawn, the  arm  dressed,  and  the  blood  gently  stirred  or  whipped  until  defibri- 
nated.  In  the  process  of  collecting  and  whipping,  the  admixture  of  air  with  the 
blood  is  to  be  avoided. 

If  only  one  generation  of  parasites  is  to  be  cultivated,  the  culture  may  be 
grown  in  the  defibrination  tube,  provided  that  the  contained  column  of  blood  be 
not  greater  than  1-2  inches.  There  is  no  advantage  in  having  a  deeper  column 
of  blood,  but  there  is  danger  in  having  less  depth  as  under  such  circumstances 
the  parasites  die  before  the  stageof  segmentation  is  reached.  In  case  the  column 
is  more  than  the  required  depth,  some  of  the  blood  can  be  pipetted  to  other  tubes 
and  several  cultures  made.  The  plasmodia  grow  in  the  top  layer  of  the  sedi- 
mented  cells,  near  the  clear  supernatant  serum  above.  The  thickness  of  the 
layer  of  cells  in  which  they  live  is  said  to  be  not  more  than  %Q  of  an  inch. 

If  the  cultures  are  to  be  continued  for  numerous  generations,  precautions 
must  be  taken  to  exempt  the  parasites  from  the  destructive  activities  of  the 
leukocytes.  The  method  is  therefore  varied  in  this  manner:  The  defibrinated 
blood  is  centrifugalized  until  three  layers  are  formed,  clear  serum  above,  leu- 
kocytes in  a  thin  layer  below,  and  red  corpuscles  at  the  bottom.  The  clear  serum 
is  pipetted  off  and  filled  into  small  culture  tubes  to  make  a  column  not  deeper 
than  1 3^  inches.  Red  blood  corpuscles  and  plasmodia  are  then  drawn  up  from 

*  Jour.  Amer.  Med.  Asso.,  1911,  LVII,  1534. 
t  "Jour.  Exp.  Med.,"  1912,  xvi,  567. 


Cultivation  of  the  Parasites  485 

the  deeper  part  of  the  corpuscular  layer,  thus  escaping  the  leukocytes  at  the  top, 
and  planted  at  the  bottom  of  each  tube  of  serum.  It  is  thought  to  be  advan- 
tageous to  use  culture  tubes  with  flat  bottoms.  A  still  better  method  is  the 
introduction  of  a  paper  disk  into  a  half-inch  tube,  about  half  an  inch  below  the 
surface  of  the  serum,  and  then  place  one-  or  two-tenths  of  a  cubic  centimeter 
of  corpuscles  upon  it.  Under  these  circumstances  all  of  the  plasmodia  are 
said  to  grow  and  segment.  Two  or  three  generations  of  parasites  grow  in  such 
cultures,  then  the  plasmodia  begin  to  die  out,  so  that  if  the  culture  is  to  be 
perpetuated,  they  must  be  transplanted  to  freshly  prepared  blood-corpuscle 
tubes  of  the  same  kind.  The  method  of  transplantation  recommended  is  so 
very  simple:  a  drop  of  the  culture  is  drawn  into  a  fine  (not  capillary)  glass 
pipette  and  then  followed  by  about  five  times  the  volume  of  the  fresh  corpuscle 
suspension.  These  are  mixed  in  the  pipette,  care  being  taken  not  to  mix  air 
with  the  blood,  and  are  then  transferred  to  the  new  media  in  the  same  manner 
as  in  making  the  original  inoculation.  The  transplantation  should  be  done  within 
five  hours  of  the  time  of  maximum  segmentation,  and  therefore  every  forty- 
eight  hours  for  the  tertian  and  aestivo-autumnal  parasites.  All  species  of  the 
plasmodia  have  been  successfully  cultivated  by  these  means.  The  parasites 
have  also  been  grown  in  red  blood-cells  in  Lock's  solution,  free  of  calcium  chlorid 
and  in  the  presence  of  ascitic  fluid. 

According  to  Bass  and  Johns,  the  parasites  grow  in  the  corpuscles,  not  upon 
them  as  believed  by  Koch.  They  are  destroyed  in  a  few  minutes  in  vitro  by 
normal  human  serum  or  by  all  the  modifications  of  it  that  they  have  tested. 
This  fact,  together  with  numerous  observations  of  parasites  in  all  stages  of  de- 
velopment apparently  within  the  corpuscles  render  untenable  the  idea  of  extra- 
corpuscular  development.  Leukocytes  phagocytize  and  destroy  malarial  para- 
sites growing  in  vitro  only  when  they  escape  from  their  red-corpuscle  capsule  or 
when  the  latter  is  perforated  or  becomes  permeable. 

The  substance  of  the  malarial  plasmodium  is  very  different  in  consistency  from 
that  of  the  blood-cells,  and  therefore  they  cannot  pass  through  the  smallest 
capillaries  like  the  more  yielding  fluid-like  red  blood-cells.  That  the  consistency 
of  the  protoplasm  of  the  parasite  is  less  yielding  than  that  of  the  red  blood-cell 
is  shown  by  the  fact  that  when  a  small  quantity  of  a  culture  containing  large 
parasites  is  spread  over  a  slide  with  the  end  of  another  slide,  the  parasites  are 
dragged  to  the  end  of  the  spread  though  the  red  blood-cells  are  left  behind. 
Large  aestivo-autumnal  plasmodia  are  round  or  oval;  the  tertian  variety  are 
more  or  less  flattened.  As  a  result  of  their  unyielding  consistency,  malarial 
parasites  lodge  in  the  capillaries  of  the  body,  especially  where  the  current  is 
weakest,  and  remain  and  segment.  In  the  meantime  other  red  corpuscles  are 
forced  against  them  and  if  in  a  favorable  situation,  one  or  more  merozoites  pass 
directly  into  the  other  cells.  When  the  segmented  parasite  has  become  suffi- 
ciently broken  up  it  can  pass  through  the  capillary  into  the  circulating  blood  where 
the  remaining  merozoites  are  almost  instantly  destroyed. 

They  further  observed  that  calcium  salts  added  to  cultures  of  aestivo-autumnal 
parasites  caused  hemolysis  of  the  infected,  possibly  also  of  non-infected  red 
blood-cells.  Such  salts  have  no  effect  on  the  corpuscles  of  normal  blood,  pos- 
sibly because  of  the  precipitation  of  other  substances  from  the  serum.  The 
amount  of  calcium  necessary  to  cause  hemolysis  of  malarial  blood  is  only  slightly 
in  excess  of  the  quantity  present  in  normal  blood  and  possibly  might  be  reached 
by  the  ingestion  of  considerable  quantities  of  calcium  in  drinking  water  or 
food.  They  speculate  that  malarial  hemoglobinuria  may  be  the  result  of  the 
presence  of  an  excess  of  calcium  in  drinking  water. 

Bass  and  Johns  believe  that  quinine  has  no  direct  effect  upon  the  malarial 
parasites,  but  effects  its  curative  influence  by  rendering  the  substance  of  the 
corpuscles  more  permeable  to  the  all-sufficient  destructive  influence  of  the 
serum.  The  quinine  would  then  affect  only  the  parasites  in  the  circulation,  and 
not  those  lodged  in  the  capillaries,  which  would  not  be  reached  until  they  had 
segmented.  The  effect  of  quinine  is  said  to  be  defeated  by  influences  such  as 
diet,  exertion,  etc.,  which  increase  the  dextrose  content  of  the  blood,  whereby 
the  permeability  of  the  red  blood-cells  seems  to  be  decreased.  It  is  hoped  that  a 
better  understanding  of  the  principles  involved  in  the  treatment  of  malaria  may 
result  from  the  study  of  the  organism  in  culture  by  which  empiricism  may  be 
exchanged  for  rationalism. 


486  Malaria 

Animal  Inoculation. — -The  human  malarial  parasites  cannot  be 
successfully  transmitted  by  experimental  inoculation  to  any  of  the 
lower  animals. 

Human  Inoculation. — The  blood  of  one  human  being  contain- 
ing schizonts,  when  experimentally  introduced  into  another  human 
being  in  doses  of  i  to  1.5  cc.  transmits  the  disease.  When  thus 
transmitted,  an  incubation  period  of  from  seven  to  fourteen  days 
intervenes  before  the  disease,  which  is  of  the  same  type  as  that  from 
which  the  blood  was  taken,  makes  its  appearance. 

Pathogenesis. — The  pathogenic  effects  wrought  by  the  malarial 
parasite  are  imperfectly  understood.  The  synchrony  of  the  seg- 
mentation of  the  parasite  with  the  occurrence  of  the  paroxysms 
seems  to  indicate  that  a  toxic  substance  saturates  and  disturbs  the 
economy  at  that  time.  Whether  it  be  an  endotoxin  liberated  by 
the  dividing  parasite  is  not,  however,  known. 

The  anemia  that  follows  infection  can  be  referred  to  the  destruc- 
tion of  the  red  blood-corpuscles  by  the  parasites  which  feed  upon 
them  and  transform  the  hemoglobin  into  melanin  (?).  When 
great  numbers  of  the  parasites  are  present  the  destruction  is  enor- 
mous, and  the  number  of  corpuscles  and  the  quantity  of  hemoglobin 
in  the  blood  sink  far  below  the  normal.  Leukopenia  instead  of 
leukocytosis  is  the  rule,  and  while  the  leukocytes  have  an  appetite 
for  the  spores  of  the  parasites  and  often  phagocyte  and  destroy  them, 
their  activity  is  not  sufficiently  rapid  or  universal  to  check  their 
rapid  increase. 

The  melanin  granules  set  free  during  sporulation  are  also  taken 
up  by  the  leukocytes  and  endothelial  cells,  the  latter  becoming 
deeply  pigmented  at  times. 

The  spleen  enlarges  as  the  disease  continues  until  it  forms  the 
" ague-cake."  The  enlargement  may  cause  the  organ  to  weigh  7 
to  10  pounds.  It  appears  to  result  from  hypertrophy.  The  tissue 
is  pigmented.  The  liver  and  kidneys  are  also  enlarged  and 
pigmented. 

Prophylaxis. — With  the  knowledge  of  the  role  of  the  mosquito 
in  the  transmission  of  malaria,  its  prophylaxis  becomes  a  matter 
of  simplicity  when  certain  measures  can  be  systematically  carried 
out.  There  are  two  equally  important  factors  to  be  considered — 
the  human  being  and  the  mosquito.  The  measures  must  be  di- 
rected toward  preventing  each  from  infecting  the  other. 

i.  The  Human  Beings. — In  districts  where  malarial  fever  pre- 
vails, the  first  part  of  the  campaign  had  perhaps  best  be  directed 
toward  finding  and  treating  all  cases  of  malarial  fever,  so  that  the 
parasites  in  their  blood  may  be  destroyed  and  the  infection  of 
mosquitoes  prevented.  This  is  done  by  the  systematic  and  general 
use  of  quinin. 

All  cases  of  malarial  fever  should  be  required  to  sleep  in  mosquito- 
proof  houses  under  nets,  and  as  the  mosquitoes  are  nocturnal  and 


Pathogenesis  487 

begin  to  fly  at  dusk,  the  patients  should  shut  themselves  in  before 
that  time.  By  thus  killing  the  parasites  in  the  blood,  and  keeping 
the  mosquitoes  from  the  patients  in  the  meantime,  much  can  be  done. 
But  where  malarial  fever  prevails,  the  mosquitoes  are  already  largely 
infected,  hence  the  healthy  population  should  also  learn  to  respect 
the  habits  of  the  insects  and  not  expose  themselves  to  their  bites, 
should  screen  their  houses  and  their  beds,  and  should  take  small 
prophylactic  doses  of  quinin  to  prevent  the  development  of  the 
parasites  when  exposure  cannot  be  avoided. 

2.  The  Mosquitoes. — It  is  not  known  that  the  parasites  can 
pass  from  one  generation  of  mosquitoes  to  another,  hence  the 
mosquitoes  to  be  feared  are  those  that  are  already  infected.  By 


Fig.  190. — Anopheles  maculipennis :  Adult  male  at  left,  female  at  right  (Howard). 

making  the  houses  mosquito-proof  most  of  the  insects  can  be  kept 
out,  while  those  that  get  in  can  be  caught  and  killed. 

By  draining  the  swamps  and  destroying  all  the  breeding  places 
in  and  near  human  habitations,  the  number  of  mosquitoes  can  be 
greatly  diminished.  Fortunately  this  is  particularly  true  with 
reference  to  the  mosquitoes  most  concerned — the  anopheles — which 
fly  but  short  distances.  By  closing  all  the  domestic  cisterns  and 
reservoirs,  cesspools,  etc.,  so  that  no  mosquitoes  can  get  in  to  breed 
or  get  out  to  bite,  and  by  draining  the  pools  for  half  a  mile  in  all 
directions  from  human  habitations,  the  number  of  anopheles  mos- 
quitoes can  be  made  almost  negligible.  If  at  the  same  time  no 
mosquitoes  are  any  longer  permitted  to  infect  themselves  by  biting 
infected  human  beings,  the  spread  of  the  disease  must  be  greatly 
restricted  or  checked. 


488 


Malaria 
MOSQUITOES  AND  MALARIAL  FEVER 


In  order  that   the   student   may  be  able   to   differentiate  with 
reasonable  accuracy  such  mosquitoes  as  come  under  his  observation, 


6  0  c 

Fig.  191. — Various  mosquitoes    in  attitudes  of   repose:   a,   Culex    pipiens;  b, 
Myzorrhynchus  pseudo-pictus;  c,  Anopheles  maculipennis    (Manson). 


Halteres 
First  abdominal  segment 


Proboscis 


Palp 

-Eye 
—  Occiput 


Prothorax 
Mesothorax 
Scutellum 
."••Metathorax 


Abdomen 


Basal  lobes 


—  First  tarsal  segment 


Fig.  192. — External   morphology  of  a  female   mosquito   (Manson). 

use  must  be  made  of  tabulations,  to  correctly  use  which,  however, 
the  student  should  have  some  familiarity  with  insect  structure  and 


Mosquitoes  and  Malarial  Fever  489 

the  general  principles  of  entomology.  The  best  works  of  reference 
for  this  purpose,  that  have  come  under  observation  to  the  present 
time  are  "A  Text-book  of  Medical  Entomology"  by  Patton  and 
Cragg,  published  by  the  Christian  Literature  Society  for  India, 
London,  Madras  and  Calcutta,  1913,  and  the  "  Handbook  of  Medical 
Entomology"  by  Riley  and  Johannsen,  the  Comstock  Publishing 
Co.,  Ithaca,  New  York,  1915. 

The  mosquitoes  comprise  a  family  of  dipterous  or  two-winged 
insects,  included  in  the  family  Culicidae.  They  can  be  recognized, 
first  by  their  well-known  general  form,  and  second  by  the  presence 
of  scales  upon  some  part  of  the  head,  thorax,  abdomen,  and  wings. 
For  the  rough  and  ready  identification  of  the  larger  groups  and 
principal  genera,  the  following  table  compiled  from  various  authors 
may  answer.  For  more  precise  information  and  for  the  identifica- 
tion of  the  species,  of  which  hundreds  are  now  described,  reference 
must  be  made  to  the  large  works  recommended  above. 
CLASSIFICATION  (Stitt) 

There  are   four  subfamilies  of  CULICIDAE,  differentiated   according  to  the 
*>alpi  : 

I.  Palpi  as  long  or  longer  than  the  proboscis  in  the  male. 

1.  Palpi  as  long  as    the  proboscis  in  the    female; 

proboscis  straight  ..........................   ANOPHELIN^E. 

2.  Palpi    as   long    or   shorter    than    the   proboscis; 

proboscis  curved  ..........  .  ................   MEGARRHININ.-E. 

3.  Palpi  shorter  than  the  proboscis  ................   CULICIN^E. 

II.  Palpi  shorter  than  the  proboscis  in  the  male  and  female  ^DIN^E. 


Of  these  the  Anophelinae  is  the  one  family  concerned  in  the  transmission  of 
malarial  fever,  so  that  it  is  important  to  be  able  to  differentiate  the  genera  in- 
cluded in  the  family. 

ANOPHELINAE 

1.  Scales  on  head  only;  hairs  on  thorax  and  abdomen. 

1.  Scales  on  wings  large  and  lanceolate.     Palpi  only 

slightly  scaled  ...............................  Anopheles. 

2.  Wing  scales  small,  narrow,  and  lanceolate.     Only  a 

few  scales  on  palpi  ............................  Myzomyia. 

3.  Large  inflated  wing  scales  .........................  Cydoleppteron. 

2.  Scales  on  head  and  thorax.     Scales  narrow  and  curved. 

Abdomen  with  hairs,  not  scales. 
i.  Wing  scales  small  and  lanceolate  ...................  Pyretophorus. 

3.  Scales  on  head,  thorax,  and  abdomen.     Palpi  covered 

with  thick  scales. 

1.  Abdominal  scales  on  ventral  surface  only.     Thoracic 

scales  like  hairs.     Palpi  rather  heavily  scaled..  .  .Myzorrhynchus. 

2.  Abdominal  scales  narrow,  curved  or  spindle  shaped, 

in  tufts  and  dorsal  patches  ....................  Nyssorrhynchus. 

3.  Abdomen  almost  completely  covered  with  scales  and 

also  having  lateral  tufts  .............  .  .........  Cellia. 

4.  Abdomen  completely  scaled  .......................  Aldrichia. 

Species  of  the  genera  Anopheles,  Myzomyia,  and  Myzorrhynchus,  are  known 
to  transmit  malarial  parasites.  The  Culicinae  include  Stegomyia  and  Culex, 
which  have  some  medical  interest,  as  the  former  transmits  yellow  fever  and  the 
latter,  filarial  worms. 

CULICINAE 

I.  Posterior  cross-vein  nearer  the  base  of  the  wing  than  the 
mid-cross-vein. 


4QO  Malaria 

1.  Proboscis  curved  in  the  female Psorophora. 

2.  Proboscis  straight  in  the  female: 

A.  Palpi  with  three  segments  in  the  female. 

a.  Third  segment  somewhat  longer  than 

the  first  two Culex. 

b.  The     three     segments     are     equal     in 

length Stegomyia. 

B.  Palpi  with  four  segments  in  the  female. 

a.  Palpi    shorter    than    the    third   of    the 

proboscis.     Spotted  wings Theobaldia. 

b.  Palpi    longer    than    the    third    of    the 

proboscis.     Irregular    scales    on    the 

wings Mansonia. 

C.  Palpi  with  fine  segments  in  the  female. .  .  .  Tceniorrhynchus. 
II.  Posterior  cross-vein  in  line  with  the  mid-cross-vein. .  .  Joblotina. 

III.  Posterior  cross-vein  further  from  the  base  of  the  wing 

than  the  mid-cross-vein Mucidus. 

Male  mosquitoes  can  at  once  be  recognized  by  the  pennate 
antennae  which  appear  like  plumes  on  each  side  of  the  head.  They 
commonly  " swarm"  in  flocks,  do  not  suck  blood,  and  are  not  com- 
monly found  in  or  about  human  habitations.  Comparatively  little 
is  known  of  their  habits.  Cohabitation  of  the  sexes  occurs  but  once 
after  which  the  males  commonly  die.  The  females  after  fecunda- 
tion require  a  meal  of  blood  before  they  become  gravid  and  ready  to 
oviposit.  Oviposition  takes  place  in  water.  During  the  winter 
many  gravid  females  hibernate  in  cellars  in  a  very  inactive  condi- 
tion, but  are  immediately  ready  to  fly  to  appropriate  places  and  lay 
their  eggs  with  the  return  of  warm  weather.  In  hot  climates  some 
of  them  estivate — i.e.,  become  similarly  inactive  during  the  dry 
period,  but  are  ready  to  fly  to  the  water  and  oviposit  as  soon  as  the 
rains  begin  again.  The  breeding  places  vary  with  the  species. 
Fresh  water  is  the  usual  preference,  but  a  few  select  pools  of  brack- 
ish water,  and  one  or  two  species  prefer  salt  water.  Most  of  the 
malaria-bear;ng  species  of  anopheles  prefer  pools  of  fresh  clear 
water,  some  prefer  running  water  in  small  streams  with  a  slow  cur- 
rent. A  few  breed  in  large  rivers.  Some  species  are  notably  domes- 
tic and  oviposite  in  wells,  cisterns,  water-butts,  cans  and  any  other 
available  collection  of  water. 

The  eggs  are  laid  as  the  female  hovers  upon  the  surface,  touching 
the  water  from  time  to  time,  with  the  tip  of  the  abdomen,  each 
time  depositing  an  egg.  Culex  eggs  are  fastened  together  side  by 
side  to  form  a  kind  of  minute  raft,  but  anopheles  eggs  are  laid  singly 
and  float  away  independently  of  one  another.  If  at  the  time  the 
waters  are  receding,  the  eggs  catch  upon  the  leaves  and  stems  of 
plants  they  may  remain  alive  until  the  waters  rise  again  before 
hatching.  Dry  eggs  are  sometimes  able  to  remain  alive  for  long 
periods,  and  may  even  be  frozen  without  being  killed.  Cazeneuve 
hatched  eight  larvae  from  eggs  obtained  by  thawing  a  block  of 
ice  taken  from  a  swamp  in  North  China,  where  the  temperature 
had  gone  as  low  as  —  32°C.  When  conditions  are  favorable  the 
eggs  hatch  in  two  or  three  weeks.  The  anopheles  larvae  feed  at 


Mosquitoes  and  Malarial  Fever 


491 


the  surface  of  the  water  along  the  banks  where  they  are  protected 
by  the  vegetation.  They  are  voracious  feeders  and  satisfy  their 
appetites  with  all  kinds  of  minute  vegetable  and  animal  organisms 
or  remnants.  In  a  day  or  two  the  larvae  molt  for  the  first  time.  In 


Fig.  193. — Pupa  of  Anopheles  maculipennis  (Brumpt). 

,    /•'  .  Brushes 

>••'...  Maxillary  palyp 

U-  ..  Antenna 

Eye 

Head 


Thora 


>  Silky  bristles 


.•  Chitinous  combs 

"•  Stigmata 
,..  Anal  papillae 
.  Large  bristles 


Abdomen 


Fig.  194. — Larva    of    Anopheles    maculipennis   (Brumpt). 

five  or  six  days,  having  grown  larger,  they  molt  a  second  time  and 
pupate.  The  appearances  of  the  larvae  and  pupae  are  shown  in  the 
accompanying  diagrams.  The  pupa  floats  at  the  surface  of  the 
water,  is  comparatively  inactive  and  does  not  feed.  If  disturbed,  it 


492 


Malaria 


is  capable  of  swimming  vigorously  to  escape.  In  about  three  days 
the  imago  issues  and  is  ready  to  fly.  Anopheles  do  not  fly  great 
distances;  a  few  hundred  yards  is  the  common  range  of  their  activi- 
ties. They  do  not  always  return  to  the  same  pools  from  which 
they  issued,  any  similar  pool  or  stream  is  good  enough  for  ovi- 


Fig.  195. — Method   of   withdrawing   the   digestive   tube  of   the   mosquito   for 

study  (Blanchard). 

position.  After  having  deposited  the  first  lot  of  eggs,  the  female 
is  ready  to  feed  again  and  produce  a  new  lot.  This  can  go  on  for 
a  number  of  broods.  How  long  the  insects  can  live,  probably  de- 
pends upon  their  activities.  When  actively  engaged  in  reproductive 
activities  they  probably  live  a  shorter  time  than  when  hibernating 


Fig.  195. — Method  of  withdrawing  the  salivary  glands  of  the  mosquito  for 

study  (Blanchard). 

or  estivating.     It  is  known  that  some  of  them  can  live  the  greater 
part  of  a  year. 

The  mosquitoes  used  for  study  and  for  classification  should  be 
mounted  dry  in  the  usual  way  well  known  to  all  entomologists. 

Fine  entomologic  pins  (oo-ooo)  should  be  employed  for  the  purpose.  The 
insects  should  be  caught  in  a  wide-mouth  bottle  containing  some  fragments 
of  cyanid  of  potassium,  covered  with  a  layer  of  sawdust,  over  which  a  thin 
layer  of  plaster  of  Paris  is  allowed  to  solidify.  The  insects  die  in  a  moment  or 
two,  can  be  emptied  upon  a  table,  and  the  pin  carefully  thrust  through  the  central 


Mosquitoes  and  Malarial  Fever  493 

part  of  the  thorax.  _  As  soon  as  the  insect  is  impaled,  the  pin  should  be  passed 
through  an  opening  in  a  card  or  between  the  blades  of  a  forceps  until  the  insect 
occupies  a  position  at  the  junction  of  the  middle  and  upper  third.  The  insect 
should  not  be  touched  with  the  fingers,  as  the  scales  will  be  brushed  off  and  the 
limbs  broken.  Mounted  insects  must  be  handled  with  entomologic  forceps, 
touching  the  pins  only.  Every  insect  thus  mounted  should  have  placed  upon  the 
pin,  at  the  junction  of  the  middle  and  lower  thirds,  a  small  bit  of  card  or  paper, 
telling  where  and  when  and  under  what  circumstances  it  was  taken. 

The  dissection  of  fresh  mosquitoes  for  determining  whether  or  not  they  are 
infected  with  malarial  organisms  must  be  made  with  the  aid  of  needles  mounted 
in  handles.  The  position  of  the  stomach,  intestines,  and  the  salivary  glands, 
and  the  mode  of  pulling  the  insect  apart  to  show  them  can  be  learned  from  the 
diagram.  The  organs  thus  withdrawn  and  separated  from  the  unnecessary 
tissue  can  be  fixed  to  a  slide  with  Meyer's  glycerin-albumin  or  other  albuminous 
matter,  and  then  stained  like  a  blood-smear,  but  should  be  cleared  after  staining 
and  washing,  and  mounted  in  Canada  balsam  under  a  cover-glass. 


Fig.  1 97- — Imago  of  Anopheles  maculipennis  escaping  from  the  pupa  case  upon 
the  surface  of  the   water    (Brumpt). 

A  more  certain  and  more  elegant  manner  of  showing  the  parasites  in  infected 
mosquitoes  is  by  pulling  off  the  legs  and  wings,  embedding  the  insect  in  paraffin 
and  cutting  serial  longitudinal  vertical  sections. 

To  infect  mosquitoes  and  study  the  development  of  the  malarial  parasites 
in  their  bodies,  the  insects  should  be  bred  from  the  aquatic  larva  in  the  laboratory, 
to  make  sure  that  they  do  not  already  harbor  parasites.  The  mosquitoes 
are  allowed  to  enter  a  small  cage  made  with  mosquito  netting,  and  are  taken  to 
the  bedside  of  the  malarial  patient,  against  whose  skin  the  cage  is  placed  until 
the  insects  have  bitten  and  distended  themselves  with  blood,  when  they  are 
taken  back  to  the  laboratory,  kept  as  many  days  as  may  be  desired,  then  killed 
and  sectioned.  In  this  way,  remembering  that  the  entire  mosquito  cycle  of  de- 
velopment takes  about  a  fortnight,  any  stage  of  the  cycle  may  be  observed. 


CHAPTER  XX 
RELAPSING  FEVER 

SPIRILLUM  OBERMEIERI   OR  SPIROCH^TA  OBERMEIERI   OR  SPIRO- 
RECURRENTIS   (OBERMEIER) 


General  Characteristics.  —  An  elongate,  flexible,  flagellated,  non-sporogenous, 
actively  motile  spiral  organism,  pathogenic  for  man  and  monkeys,  susceptible 
of  cultivation  in  special  media,  stained  by  ordinary  methods,  but  not  by 
Gram's  method. 

IN  1868  Obermeier*  first  observed  the  presence  of  actively  motile 
spiral  organisms  in  the  blood  of  a  patient  suffering  from  relapsing 
fever.  Having  made  the  observation,  he  continued  to  study  the 
organism  until  1873,  when  he  made  his  first  publication.  From  1873 
until  1890  it  was  supposed  that  spirochseta  rarely  played  any  patho- 
genic role.  Millerf  had,  indeed,  called  attention  to  the  constant 
presence  of  Spirochseta  dentinum  in  the  human  mouth,  but  it  had 
not  been  connected  with  any  morbid  condition.  In  1890  Sacharofff 
discovered  a  spirillary  infection  of  geese  in  the  Caucasus,  caused  by 
an  organism  much  resembling  Spirochaeta  obermeieri  and  called 
Spirochaeta  anserinum.  In  1903  Marchoux  and  Salimbeni§  found 
a  third  disease,  fatal  to  chickens,  caused  by  Spirochaeta  gallinarum, 
and  found  that  the  spread  of  the  disease  was  determined  by  the 
bites  of  a  tick,  Argas  miniatus.  In  1902  Theiler,||  in  the  Transvaal, 
observed  a  spiral  organism  in  a  cattle  plague.  This  has  been  named 
after  him  by  Laveran,  Spirochaeta  theileri.  It  was  found  to  be 
disseminated  by  the  bites  of  certain  ticks  —  Rhipicephalus  decolor- 
atus.  Later,  what  was  probably  the  same  organism,  was  found  in 
the  blood  of  sheep  and  horses.  In  1905  Nicolle  and  Comte**  found 
a  spiral  organism  infecting  certain  bats.  By  this  time,  therefore, 
it  became  evident  that  spirochaetal  infections  were  fairly  well  dis- 
seminated among  the  lower  animals  and  that  the  spirochaeta  were 
of  different  species  with  different  hosts  and  intermediate  hosts. 

In  1904  Ross  and  Milnett  and  Button  and  Todd||  studied  a 
peculiar  African  fever  which  they  were  able  to  refer  to  a  spirochaeta 

*"Centralbl.  f.  d.  med.  Wissenschaft,"   1873. 

f  Microorganisms  of  the  Human  Mouth,  Phila.,  1890,  p.  44  et  seq. 

j"Ann.  de  PInst.  Pasteur,"  1891,  xvi,  No.  9,  p.  564. 

§  Ibid.,  1903,  xvn,  p.  569. 

||  "  Jour.  Comp.  Path,  and  Therap.,"  1903,  XLVII,  p.  55. 
**  "  Compt.-rendu  de  la  Soc.  de  Biol.  de  Paris,"  July  22,  1905,  LIX,  p.  200. 
ft  "British  Med.  Jour.,"  Nov.  26,  1904,  p.  1453. 

tj  "Memoir  xvn,  Liverpool  School  of  Tropical  Medicine,"  "Brit.  Med. 
Jour.,"  Nov.  u,  1905,  p.  1259. 

494 


Relapsing  Fever  495 

for  which  Novy*  has  proposed  the  name  Spirochaeta  duttoni  in 
memory  of  Button,  who  lost  his  life  while  studying  it.  In  1905 
Kochf  while  working  in  Africa  discovered  a  spirochaeta  that  he  re- 
garded as  identical  with  that  already  described  by  Ross  and  Milne 
and  Button  and  Todd.  Later  studies  of  the  organism  convinced 
C.  Frankel  J  that  it  was  a  separate  species.  For  it  Novy  later  sug- 
gested the  name  Spirochaeta  kochi.  In  1906  Norris,  Pappenheimer 
and  Flournoy §  found  a  spirochaeta  in  the  blood  of  a  patient  suffering 
from  relapsing  fever  in  New  York.  This  having  been  extensively 
studied  by  Novy,  has  since  been  called  Spirochaeta  novyi. 

With  the  work  of  Schaudinn  and  his  associate,  Hoffmann,  || 
the  spirochaeta  came  to  be  regarded  as  protozoan  parasites  because 
of  the  presence  of  an  undulating  membrane;  the  refusal  of  most  of 
the  organisms  to  grow  upon  artificial  media,  the  role  of  an  inter- 
mediate host  (ticks,  etc.)  in  transmitting  them,  and  the  longitudinal 
mode  of  division. 

Fevers  characterized  by  relapses  and  by  the  presence  of  spiro- 


Fig.  198. — Spirochseta  obermeieri  from  human  blood  (Kolle  and  Wassermann) . 

chaeta  in  the  blood  have  been  found  in  northern  and  northeastern 
Europe  (true  relapsing  fever  with  Spirochaeta  obermeieri),  in  various 
parts  of  equatorial  Africa  (African  relapsing  fever  with  Spirochaeta 
duttoni) ;  in  North  Africa  (Spirochaeta  berbera) ;  in  Bombay  and  in 
other  parts  of  India  (Spirochaeta  carteri);  in  Persia  (Spirochaeta 
persica) ;  and  in  America  (Spirochaeta  novyi).  The  question,  there- 
fore, arises  whether  these  similar  diseases  are  slight  modifications 

*"Jour.  Infectious  Diseases,"  1906,  in,  p.  295. 

f'Deutsche  med.  Wochenschrift,"  1905,  xxxi,  p.  1865;  "Berliner  klinische 
Wochenschrift,"  1906,  XLIII,  185. 

t  "Med.klin.,"  1907,111,928;  "Miinchener  med.  Wochenschrift,"  1907, LIV,  201. 

§"Jour.  Infectious  Diseases,"  1906,  m,  266. 

||  "Deutsche  med.  Wochenschrift,"  Oct.,  1905,  xxxi,  p.  1665;  "Arbeiten  aus 
dem  kaiserlichen  Gesundheitsamte,"  1904,  xx,  pp.  387—439. 


49^  Relapsing  Fever 

of  the  same  thing  caused  by  the  same  parasite,  or  whether  they 
are  different  diseases  caused  by  slightly  different  parasites. 

If  Nuttall  be  correct,  there  are  no  adequate  grounds  upon  which 
to  conclude  that  the  spirochetes  are  really  different  species.  On 
this  account,  and  as  the  differences  between  the  organisms  are 
minute,  it  scarcely  seems  well  to  devote  space  to  the  consideration 
of  each,  but  better  to  select  the  oldest  and  the  best  known — Spiro- 
chaeta  obermeieri— as  the  type,  describe  it,  and  then  point  out  such 
variations  as  are  shown  by  its  close  relations. 

Morphology. — The  Spirochaeta  obermeieri  is  extremely  slender, 
flexible,  spirally  coiled,  like  a  corkscrew,  and  pointed  at  the  ends. 


Fig.  199. — Spirochaeta   obermeieri    (Novy).     Rat   blood   No.   32ia.     X   1500. 

It  measures  approximately  i  n  in  breadth  and  10,  20,  or  even  40  ^ 
in  length.  The  number  of  spiral  coils  varies  from  6  to  20;  the  di- 
ameter of  the  coils  varies  so  greatly  that  scarcely  any  two  are  uni- 
form. Wladimiroff  *  doubts  the  existence  of  a  flagellum,  but  flagella- 
like  appendages  are  usually  to  be  seen  at  one  or  both  ends  of  the 
organisms.  An  undulating  membrane  attached  nearly  the  entire 
length  of  the  organism,  very  narrow,  and  inconspicuous,  forms  the 
chief  means  of  locomotion.  The  organism  is  actively  motile, 
and  darts  about  in  fresh  blood  with  a  double  movement,  consisting 
of  rotation  about  the  long  axis  and  serpentine  flexions.  No  structure 
can  be  made  out  by  our  present  methods  of  staining  and  examining 
the  Spirochaeta.  No  spores  are  found.  Multiplication  is  thought 
to  take  place  by  longitudinal  division,  though  some  believe  the  di- 
vision to  be  transverse. 

"Kolle   and   Wassermann's   Handbuch   der  pathogene  Mikroorganismen," 
1903,  in,  p.  82. 


Cultivation  497 

The  Spirochaeta  duttoni  is  said  by  Koch,*  in  his  interesting 
studies  of  "  African  Relapsing  Fever,"  to  resemble  the  Spirochaeta 
obermeieri  in  all  particulars. 

The  Spirochaeta  novyi  with  which  Novy  and  Knappf  experi- 
mented, and  which  they  believed  to  be  identical  with  Spirochaeta 
obermeieri,  measured  0.25  to  0.3  JJL  in  breadth  by  7  to  19  n,  in  length. 
The  number  of  coils  varies  from  three  to  six.  The  shorter  forms  are 
pointed,  with  a  long  flagellum  at  one  end  and  a  short  one  at  the 
other. 

Staining. — The  Spirochaeta  can  be  stained  with  ordinary  anilin 
dye  solutions,  by  the  Romanowsky  and  Giemsa  methods,  and  by 
the  silver  methods  (see  Treponema  pallidum).  It  does  not  stain 
by  Gram's  method. 


Fig.  200. — Spirochaeta    duttoni    (Novy).     Tick    fever,    No.    520.     Rat  blood. 

X  1500. 

Cultivation. — -Following  the  suggestion  of  Levaditi,  Novy  and 
Knappt  cultivated  Spirochaeta  obermeieri  in  collodion  sacs  in  the 
abdominal  cavity  of  rats,  and  succeeded  in  maintaining  it  alive  in 
this  way  through  twenty  consecutive  passages  during  sixty-eight 
days.  They  were  able  to  do  this  in  rat  serum  from  which  all  cor- 
puscles had  been  removed  by  %  centrif ugation,  and  so  proved  that 
no  intercellular  developmental  stage  of  the  organism  takes  place. 
Organisms  thus  cultivated  attenuate  in  virulence. 

Norris,  Pappenheimer,  and  Flournoy§  believe  that  they  succeeded 
in  securing  multiplication  of  the  Spirochaeta  by  placing  several  drops 

"Berliner  klin.  Wochenschrift,"  Feb.  12,  1906,  xxxrv,  No.  7,  p.  185. 
'  "Jour.  Infectious  Diseases,"  1906,  in,  p.  291. 
j  "Jour.  Amer.  Med.  Assoc.,"  Dec.  29,  1906,  XLVII,  p.  2152. 
§  "Journal  of  Infectious  Diseases,"  1906,  HI,  266. 
32 


498  Relapsing  Fever 

of  blood  containing  them  in  3  to  5  cc.  of  citrated  rat  or  human 
blood.  A  third  generation  always  failed. 

Noguchi*  was  the  first  to  achieve  the  successful  cultivation  of 
the  spirochaeta  in  artificial  culture  media.  The  best  success  was 
obtained  as  follows:  Into  each  of  a  number  of  sterile  test-tubes 
2  X  20  cm.  in  size  is  placed  a  fragment  of  fresh  sterile  rabbit  kidney 
and  then  a  few  drops  of  citrated  blood  from  the  heart  of  an  infected 
mouse  or  rat.  Following  this,  about  15  cm.  of  sterile  ascitic  or 
hydrocele  fluid  are  quickly  poured  into  the  tubes  and  the  contents 
of  some  of  the  tubes  are  covered  with  a  layer  of  sterile  parafnne 
oil,  while  the  rest  are  left  without  the  oil.  The  tubes  are  placed  in 
the  incubating  oven  at  37°C.  By  these  means  cultures  of  Spiro- 
chaeta duttoni,  Spirochaeta  kochi,  Spirochaeta  obermeieri  and  Spiro- 
chaeta novyi  were  secured.  The  maximum  growth  was  obtained  in 
7,  8  or  9  days  at  37°C.  The  presence  of  some  oxygen  seemed  to  be 
essential.  By  transplantations  to  fresh  media  of  the  same  kind 
they  were  all  kept  growing  for  many  generations  during  which  they 
did  not  lose  their  virulence. 

Mode  of  Infection. — The  means  by  which  Spirochaeta  obermeieri 
is  transmitted  from  individual  to  individual  is  not  definitely  known. 
Tictinf  seems  to  have  been  the  first  to  believe  that  the  transmission 
of  the  disease  was  accomplished  through  the  intermediation  of  some 
blood-sucking  insect.  He  investigated  lice,  fleas,  and  bed-bugs, 
in  the  latter  of  which  he  was  able  to  find  the  organisms,  and  through 
blood  obtained  from  which  he  was  able  to  transmit  the  disease  to 
an  ape.  He  was  not  able  to  infect  apes  by  permitting  infected 
bed-bugs  to  bite  them.  Breinl  and  Kinghorn  and  Toddf  made  a 
careful  study  of  the  subject,  but,  like  Tictin  and  their  other  prede- 
cessors, were  unable  to  infect  monkeys  by  permitting  infected  bed- 
bugs to  bite  them. 

Mackie,§  Graham-Smith,  ||  Bousfield,**Ed.SergentandH.Foley,tt 
studied  the  louse  and  found  that  it  was  undoubtedly  capable  of 
acting  as  a  transmitting  agent,  and  possibly  was  the  only  definitive 
host  of  the  parasite.  Nicolle,  Blaizot  and  Consent J  studied  the 
North  African  relapsing  fever  of  Tunis  and  Algeria,  and  proved  that 
the  body  and  head  lice  are  undoubtedly  the  common  definition  hosts 
of  its  spirochaete.  When  the  lice  were  fed  upon  blood  of  infected 
patients,  the  spirochaetes  rapidly  disappear  in  their  bodies,  but  after 
eight  days  reappear  and  remain  for  almost  twelve  days  during  which 
time  the  insects  can  transmit  the  disease.  They  also  found  that  the 

*  "Journal  of  Experimental  Medicine,"  1912,  xvi,  199. 

t  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1894,  i  Abt,  xv,  p.  840. 

j  Ibid.,  Oct.,  1906,  XLII,  Heft  6,  p.  537. 

§  "Brit.  Med.  Jour.,"  Dec.  14,  1907. 

||  "Ann.  de  PInst.  Pasteur,"  1910,  p.  63. 

!*  Report  of  the  Wellcome  Tropical  Research  Laboratories,  1911,  p.  63. 

•  "'Ann.  de  PInst.  Pasteur,"  1910,  p.  337. 
It  "Ann.  de  PInst.  Pasteur,"  Mar.  25,  1913,  vol.  xxvii,  No.  3,  p.  204. 


Mode  of  Infection  499 

infectious  agent  passes  to  a  new  generation  of  the  lice,  which  are 
also  infective.  They  also  studied  a  tick,  Ornithodorus  savignyi, 
found  in  those  countries,  thinking  that  it  might  behave  like  Ornith- 
odorus moubata  toward  Spirochaeta  duttoni,  and  found  that  it 
could  transmit  the  spirochaete  of  the  Tripolitan  relapsing  fever, 
though  apparently  not  that  of  the  Tunisian  fever. 

When  we  come  to  consider  Spirochaeta  duttoni,  however,  we  find 
our  knowledge  much  further  advanced.  On  Nov.  26,  1904,  Dutton 
and  Todd  announced  that  they  had  discovered  a  spirillum  to  be 
the  specific  agent  in  the  causation  of  tick  fever  in  the  Congo,  and  on 
the  same  date  Ross  and  Milne*  published  the  same  fact.  Dutton 
and  Todd  subsequently  withdrew  their  claim  to  priority  of  the 
discovery.  On  Feb.  4,  1905,  Ross  published  in  the  "  British  Medical 
Journal"  the  following  cablegram  from  Dutton  and  Todd,  then 
working  on  the  Congo:  " Spirilla  cause  human  tick  fever;  naturally 
infected  ornithodorus  infect  monkey."  It  was  not  until  Nov.  n, 
1905,  that  the  paper  upon  the  subject  was  read  and  published  in 
the  same  journal  by  Dutton  and  Todd,  and  the  etiology  of  the  dis- 
ease made  clear.  These  observers  found  that  the  horse-tick, 
Ornithodorus  moubata  (Murray)  is  the  intermediate  host  of  the 
spirilla  or  spirochaeta  causing  the  disease,  and  that  when  these  ticks 
were  permitted  to  bite  infected  human  beings,  and  then  subsequently 
transferred  to  monkeys,  the  latter  sickened  with  the  typical  infection. 

The  matter  received  confirmation  and  addition  through  the  studies 
of  Koch,f  who  studied  the  ticks,  observed  the  distribution  of  the 
micro-organisms  in  their  bodies,  and  found  that  they  collected  in 
large  numbers  in  the  ovaries,  so  that  the  eggs  were  commonly  in- 
fected and  the  embryo  hexapod  ticks  hatched  from  them  were  in- 
fective. Not  only  is  this  second  generation  of  ticks  infected,  but 
Moller  has  found  the  third  generation  also  infected  by  the  spiro- 
chaeta,  and  it  is  not  improbable  that  the  infection  is  kept  on  passing 
from  female  to  offspring  through  many  generations.  Leishman, 
who  followed  the  spirochaeta  throughout  the  body  of  the  tick, 
observed  that  it  entered  the  ovaries  and  appeared  in  the  ova  in  the 
spiral  form,  but  that  in  the  ova  it  not  infrequently  became  trans- 
formed to  "coccoid"  granules  which  held  together  more  or  less 
closely  like  tiny  streptococci.  He  supposed  that  it  was  in  the 
granular  form  that  the  micro-organism  found  its  way  into  the  embryo 
and  so  infected  the  developing  nymph.  There  is  reason  to  believe 
that  this  was  an  error  and  that  the  spirals  alone  are  the  sources  of 
transmission  and  infection.  What  is  true  of  the  tick  seems  to  be 
equally  true  of  the  lice,  the  infective  micro-organisms  being  passed 
down  from  generation  to  generation.  Thus,  in  regard  to  Spirochaeta 
duttoni  we  are  able  to  say  quite  definitely  that  the  tick  is  the  usual 
if  not  the  only  means  of  dissemination.  How  the  ticks  and  lice 

*  "British  Medical  Journal,"  Nov.  26,  1904. 
t"  Berliner  klin.  Wochenschrift,"  Feb.  12,  1906. 


500  Relapsing  Fever 

effect  the  transmission  of  micro-parasites  is  to  a  certain  extent  in 
dispute.  It  was  at  first  supposed  that  the  spirochaetes  entered  the 
human  hosts  with  the  saliva  of  the  respective  arthropods,  but  there 
is  some  reason  to  think  that  this  is  a  mistake,  and  that  the  scratch- 
ing of  the  itching  bite  conveys  the  spirochaeta  deposited  upon  the 
skin  in  the  excrement  of  the  arthropod,  into  the  deeper  layers  and 
lymphatics  through  which  it  reaches  the  blood. 

Pathogenesis. — 'The  spirochaeta  of  relapsing  fever  are  pathogenic 
for  man  and  monkeys,  some  of  them  for  smaller  animals.  Novy 
and  Knapp*  found  their  organism  and  Spirochaeta  duttoni  to  be 
infectious  for  mice  and  rats,  and  attribute  the  failure  of  others  to 
discover  this  to  their  failure  to  examine  the  blood  during  the  first 
and  second  days.  Fulleborn  and  Meyer,  and  Martinf  were  able 
successfuly  to  transmit  .the  spirochaeta  of  Russian  relapsing  fever 
to  mice  after  first  passing  it  through  apes.  Rabbits  and  guinea- 
pigs  seem  to  be  refractory;  white  mice  susceptible.  Man,  monkeys, 
and  mice  suffer  from  infection  characterized  by  relapses,  and  in 
them  the  disease  may  be  fatal.  Rats  never  die  of  the  disease  and 
rarely  have  relapses. 

The  micro-organisms  are  free  parasites  of  the  blood  in  which 
they  swim  with  a  varying  rapidity,  according  to  the  stage  of  the 
disease.  They  are  present  during  the  febrile  paroxysms  only, 
disappearing  completely  as  soon  as  the  crisis  is  reached. 

The  course  of  relapsing  fever  in  man  is  peculiar  and  characteristic. 
After  a  short  incubation  period  the  invasion  comes  on  with  chill, 
fever,  headache,  pain  in  the  back,  nausea  and  vomiting,  and  some- 
times convulsions.  The  temperature  rises  rapidly  and  there  are 
frequent  sweats.  The  pulse  is  rapid.  By  the  second  day  the  tem- 
perature may  be  104°  to  io5°F.  and  the  pulse  no  to  130.  There 
is  enlargement  of  the  spleen.  Icteroid  discoloration  of  the  conjunc- 
tiva may  be  observed.  The  fever  persists  with  severity  and  the 
patient  appears  very  ill  for  five  or  six  days,  when  a  crisis  occurs, 
and  the  temperature  returns  to  normal;  there  is  profuse  sweating 
and  sometimes  marked  diarrhea,  and  the  patient  at  once  begins  to 
improve.  So  rapid  is  the  convalescence  that  in  a  few  days  he  may 
be  up  and  may  desire  to  go  out.  The  disease  is,  however,  not  at 
an  end,  for  on  or  about  the  fourteenth  day  the  relapse  characteristic 
of  the  affection  makes  its  appearance  as  an  exact  repetition  of  what 
has  gone  before.  This  is  followed  by  another  apyretic  interval, 
and  then  by  another  relapse,  and  so  on.  The  patient  usually  re- 
covers, the  mortality  being  about  4  per  cent.  The  fatal  cases 
are  usually  old  or  already  infirm  patients.  The  Indian,  African, 
and  American  varieties  present  variations  of  no  great  importance. 
The  European  fever  usually  ends  after  the  second  or  third  relapse, 
the  African  not  until  after  a  greater  number. 

*  Loc.  cit.  t  Loc.  cit. 


The  Vectors  of  Relapsing  Fever  501 

The  spirochaeta  are  present  in  the  blood  in  great  numbers  during 
the  febrile  stages,  but  entirely  disappear  during  the  intervals. 

Lesions. — 'There  are  no  lesions  characteristic  of  relapsing  fever. 

Bacteriologic  Diagnosis. — -This  should  be  quite  easily  made  by 
an  examination  of  either  the  fresh  or  stained  blood,  provided  the 
blood  be  secured  during  a  febrile  paroxysm.  The  readiness  with 
which  the  organisms  take  the  stain  leaves  little  to  be  desired. 

Novy  and  Knapp  have  found  that  the  serum  of  recovered  cases 
can  be  used  to  assist  in  making  diagnosis  because  of  its  agglutinating, 
germicidal,  and  immunizing  powers. 

Immunity. — 'The  phenomena  of  immunity  are  vivid  and  im- 
portant. At  the  moment  of  decline  of  the  fever  a  powerful  bacterio- 
lytic  substance  appears  in  the  blood  and  dissolves  the  organisms. 
At  the  same  time  an  immunizing  substance  appears.  The  two  do 
not  appear  to  be  the  same. 

The  immunizing  body  affords  future  protection  to  the  individual 
for  an  indefinite  length  of  time.  It  can  be  increased  by  rapidly  in- 
jecting the  animal  with  blood  containing  spirochaeta.  Serum  con- 
taining the  immunizing  body  imparts  passive  immunity  to  other 
animals  into  which  it  is  injected,  and,  according  to  Novy  and  Knapp, 
establishes  a  solid  basis  for  the  prevention  and  cure  of  relapsing 
fever  in  man. 

THE  VECTORS  OF  RELAPSING  FEVER 
I.  TICKS 

The  ticks  thus  far  known  to  act  as  vectors  of  relapsing  fever  are  two  species  of 
the  genus  Ornithodorus.  Thirteen  species  of  this  genus  are  described  in  "A 
Text-book  of  Medical  Entomology,"  by  Patton  and  Cragg,  who  give  excellent 
tables  for  their  identification  and  additional  valuable  information  is  to  be  found 
in  the  excellent  "Monograph  of  the  Ixodoidea,"  by  Nuttall.  Ornithodorus 
ticks  of  various  species  are  to  be  found  pretty  widely  distributed  throughout 
tropical  and  semitropical  regions  of  both  hemispheres.  ^  In  general,  they  are  most 
numerous  where  the  temperature  is  highest  and  the  soil  driest. 

The  genus  Ornithodorus  was  described  by  C.  L.  Koch  and  characterized  as 
follows:  "The  body  is  flat  when  starving  and  convex  when  replete,  and  may  be 
nearly  as  broad  anteriorly  as  posteriorly,  or  pointed  and  beak-like  anteriorly. 
The  margin  of  the  body  is  not  distinct  but  is  of  a  similar  structure  to  the  rest  of 
the  integument  which  is  generally  mamillated.  On  the  ventral  surface  there 
are  two  welt-marked  folds,  one  internal  to  the  coxae,  the  coxal  fold,  and  the  other 
above  the  coxae,  the  supracoxal  fold;  there  is  also  a  transverse  pre-anal  groove, 
as  well  as  a  transverse  postanal  groove.  Eyes  are  either  absent  or  present  in 
pairs  on  the  supracoxal  fold;  one  pair  between  coxae  I  and  II,  and  the  other 
between  coxae  III  and  IV. 

The  Ornithodorus  savignyi  is  the  transmitting  agent  of  Spirochaeta  berbera; 
Ornithodorus  moubata  of  Spirochaeta  duttoni. 

Ornithodorus  savignyi. — The  description  given  by  Patton  and  Cragg  ("A  Text- 
book of  Medical  Entomology,"  1913,  p.  586)  is  as  follows:  Integument  leathery 
and  covered  by  distinct  non-contiguous  mammillae  and  numerous  short  hairs 
interspersed.  Supracoxal  folds  well  marked,  with  two  eyes  on  each  side. 
Coxal  folds  less  well  marked.  Pre-anal  groove  distinct.  The  basis  capituli 
broader  than  long  and  shorter  than  the  rest  of  the  rostrum.  Hypostome  with 
six  principal  rows  of  teeth,  the  external  the  stoutest.  Palps  with  first  and  second 
segments  of  equal  length,  third  segment  the  shortest.  Coxae  contiguous;  pro- 
tarsus  and  tarsus  of  legs  I,  II  and  III  with  three  well-marked  humps;  the  two 
proximal  humps  on  tarsus  of  leg  IV  are  close  to  each  other,  while  the  third  is 


502 


Relapsing  Fever 


separated  by  an  interval  of  about  two  and  a  half  times  the  distance  between  the 
first  and  second. 

Length  5-12  mm.  Width  4-8.5  mm.  The  female  and  male  resemble  each 
other  except  that  the  latter  are  smaller.  Its  genital  orifice  is  markedly  smaller. 
In  the  female  the  genital  orifice  is  a  broad  transverse  slit  which  can  be  made 
to  gape  and  is  guarded  by  two  flaps  like  valves;  in  the  male  the  orifice  is  oval  and 
the  valves  are  absent.  The  eggs  number  50-100,  measure  1.3-1.5  mm.  in  length 
and  0.8-1  mm.  in  breadth.  They  are  oval,  smooth  and  of  a  dark  brown  or  black 
color. 


Fig.  201. — Ornithodorus  moubata.     Tick  that  transmits  African  relapsing 
fever:  a,  Viewed  from  above;  b,  viewed  from  below  (Murray  from  Doflein). 


Fig.  202. — Ornithodorus  savignyi.  An,  anus;  cam,  camerostome;  cx.I,  coxa 
I;  ex. II,  coxa  II;  cx.III,  coxa  III;  ex. IV,  coxa  IV;  cx.f.,  coxal  fold;  e,  eye;  g.a., 
genital  aperture;  g.g.,  genital  groove. 

Habitat. — Arabia,  Nubia,  Egypt,  Somaliland,  Abyssinia,  German  East  Africa, 
Cape  Colony,  Rhodesia,  Bechuanaland  and  Portuguese  East  Africa.  In  India  it 
is  common  in  the  Madras  Presidency,  in  Gujarat,  and  in  many  parts  of  the 
Bombay  Presidency.  In  Aden  it  is  widely  distributed  throughout  the  Hinter- 
land, where  its  principal  host  is  the  camel. 

Ornithodorus  moubata. — Patton  and  Cragg  describe  this  tick  as  follows: 
Body  almost  as  broad  anteriorly  as  posteriorly;  covered  with  non-contiguous 
mamillEe,  but  with  fewer  hairs  than  savignyi.  Basis  capituli  broader  than  long 
and  shorter  than  the  palps;  hypostome  withsix  principal  rows  of  teeth.  Tarsi  of 
legs  I,  II  and  III  with  three  humps  as  in  savignyi;  those  on  the  pro-tarsus  are 


The  Vectors  of  Relapsing  Fever 

Female  Male 


503 


Ovum  or  nit  Embryo 

Fig.  203. — Pediculus  capitis,  or  head-louse.  X  10.  a,  Female;  b,  male;  c,  egg 
cemented  to  a  hair;  d,  nymph.  (From  Beattie  and  Dickson's  "A  Text-book  of 
General  Pathology,"  by  kind  permission  of  William  Heinemann,  Publisher.). 


Male 


Female 


Embryo  Ovum 

Fig.  204.— Pediculus  vestimenti,  the  clothes  or  body  louse.  X  10.  a,  Male, 
b  female;  c,  nymph;  d,  egg.  (From  Beattie  and  Dickson's  "  A  Text-book  of 
General  Pathology,"  by  kind  permission  of  William  Heinemann,  Publisher.) 


504  Relapsing  Fever 

subequal,  more  pointed  and  about  equidistant,  while  those  of  savignyi  are 
unequal,  less  pointed  and  not  equidistant.  The  tarsus  of  leg  IV  in  moubata  is 
shorter  and  thicker  than  in  savignyi,  and  its  humps  are  nearly  equidistant. 
Eyes  absent.  Length  8-12  mm.;  breadth  6-10  mm.  The  eggs  are  ovoid,  meas- 
ure 0.8-0.9  mm.  in  length,  are  smooth  on  the  surface  and  dark  yellow  in  color. 

Habitat. — Africa:  from  British  East  Africa  to  the  Transvaal,  and  across  to  the 
Congo;  southward  to  German  East  Africa  and  Cape  Colony.  It  is  common  in 
Egypt,  Abyssinia  and  in  parts  of  Somaliland  and  in  Portuguese  East  Africa. 

Ornithodorus  savignyi  is  chiefly  a  parasite  of  the  camel  and  only  occasionally 
bites  man;  Ornithodorus  moubata  is  essentially  a  human  pest. 

The  eggs  of  these  ticks  hatch  in  eight  to  fourteen  days.  The  larval  stage  which 
has  sixlegsis  spent  in  the  eggs  and  the  creature  that  emerges  is  usually  a  first  nym- 
phalinston,  which  has  eight  legs.  After  hatching  it  remains  inactive  for  several 
days,  then  becomes  very  active  and  ready  to  suck  blood.  As  it  grows  it  becomes 
voracious,  distending  itself  with  blood,  then  dropping  off,  hiding  itself  for  a  time, 


I 


Male  Female 

Fig.  205. — Pediculus  pubis,  Phthirius  inguinalis  or  crab-louse.  X  17.  (From 
Beattie  and  Dickson's  "A  Text-book  of  General  Pathology,"  by  kind  permission 
of  William  Heinemann,  Publisher.) 

molting,  then  being  ready  to  feed  again.  This  continues  for  a  number  of 
months,  the  ticks  molting  four  times  before  passing  from  the  nymph  to  the  adult 
stage.  ^ 

Ornithodorus  moubata  is  a  common  inhabitant  of  the  native  African  huts  along 
the  caravan  routes.  To  avoid  it  and  escape  relapsing  fever  R.  Koch  in  his 
African  expedition  camped  near  but  not  in  the  villages,  and  avoided  the  native 
houses.  It  lives  in  the  cracks  in  the  mud  walls,  in  the  thatch,  in  the  mats  and 
sometimes  simply  upon  the  ground  where  its  small  size  and  dull  color  make  it 
difficult  to  see.  From  these  hiding  places  it  crawls  at  night  and  like  abed-bug 
attacks  the  sleeping  host.  When  handled  it  feigns  death,  remaining  quiet  for 
so  long  a  time  that  it  is  hard  to  believe  it  alive. 

The  Ornithodorus  savignyi  is  less  adapted  to  the  requirements  of  the  spiro- 
chseta  than  its  relative.  Brumpt*  found  that  the  spirochaeta  did  not  pass 
through  the  eggs  of  O.  savigyni  to  subsequent  generations,  and  that  the  in- 
fectivity  of  the  tick  itself  soon  was  lost.  The  spirochaetae  remain  indefinitely 
in  O.  moubata,  and  are  passed  through  their  eggs  to  at  least  three  generations. 
It  is,  therefore,  difficult  to  be  certain  that  any  particular  tick  is  uninfected  unless 
its  progenitors  be  known. 

The  spirochaeta  pass  from  female  to  the  ovum  and  infect  the  young  nymphs  as 
such.  The  granules  observed  in  the  eggs  of  infected  ticks,  also  occur  in  those  of 
non-infected  ticks  and  have  nothing  to  do  with  the  spirochaeta. 

*"  Precis  de  Parasitologie,"  1910,  538. 


The  Vectors  of  Relapsing  Fever  505 

II.  LICE 

Lice  are  apterous  insects  formerly  classed  in  the  order  Hemiptera,  but  now 
placed  in  a  separate  order,  the  Anoplura.  Two  genera,  and  three  species  are 
common  upon  human  beings. 

I.  Pediculus  (Linn,  1758).     In  this  genus  there  are  two  species: 

1.  Pediculus  capitis  (de  Geer,  1778).     This  is  the  head-louse.     It  is  of  a 

gray  color.  The  abdomen  is  composed  of  eight  and  not  of  seven 
segments  as  was  stated  by  Piaget,  and  is  blackened  along  the  edges. 
The  males  and  females  look  much  alike,  but  the  male  measures  1.8  mm. 
in  length  and  0.7  mm.  in  breadth,  while  the  female  measures  2.7  mm. 
in  length  by  i  mm.  in  breadth. 

These  parasites  live  in  the  hair,  close  to  the  scalp.  Rarely  they 
pass  from  the  scalp  to  the  beard.  Still  more  rarely  do  they  occur 
upon  other  hair-covered  surfaces.  The  female  produces  large  eggs, 
one  at  a  time,  which  are  firmly  anchored  to  the  hairs  by  a  mucilaginous 
secretion.  In  them  the  embryo  develops  in  about  sixteen  to  eighteen 
days  then  escapes  as  a  nymph  with  proportionally  smaller  body  and 
larger  legs  than  the  adult.  There  are  three  molts  before  the  insect 
reaches  maturity.  The  full  and  empty  eggs  occur  in  great  numbers 
upon  the  hairs  and  are  known  as  "nits." 

The  insects  are  sometimes  present  on  the  head  in  great  numbers  and 
cause  intolerable  itching. 

2.  Pediculus  vestimenti  (Nitzsch,  1818).     This  is  a  larger  louse  of  much  the 

same  appearance  and  structure  as  P.  capitis.  Indeed  there  are  such 
minute  differences  between  the  two  that  there  is  some  dispute  as  to 
whether  they  should  not  form  subspecies  of  the  same  insect  instead 
of  different  species  of  insects. 

The  size  is,  however,  larger.  The  male  measures  3  mm.  in  length 
and  i  mm.  in  breadth;  the  female  3.3  mm.  in  length  and  1.14  in 
breadth. 

The  "body  louse"  as  this  is  commonly  called,  lives  in  the  clothing, 

and  passes  to  the  skin  to  feed,  then  returns  again  to  the  seams  of  the  garments. 

Its  eggs  are  fastened  to  the  fabric  of  the  clothing,  not  to  the  skin  or  hairs.     It  is 

sometimes  present  in  great  numbers  and  its  bites  cause  much  annoying  itching. 
Both  of  these  lice  have  been  found  to  be  capable  of  effecting  the  transmission 

of  the  spirochaeta  of  relapsing  fever.     The  infection  in  the  lice  is  transmitted  to 

its  offspring  as  in  the  case  of  Ornithodorus  moubata. 

II.  Phthirius  (Leach,  1815).  In  this  genus  there  is  only  one  human  parasite. 
Phthirius  inguinalis  (Ridi,  1668).  This  pubic  louse  or  "crab  louse  "  is 
often  incorrectly  called  Pediculus  pubis.  It  is  a  shorter,  stouter- 
bodied  creature  with  more  powerful  legs  terminating  in  large  tarsal 
hooks  that  give  it  a  crab-like  appearance.  The  thorax  and  abdomen 
are  compressed  and  shortened  to  a  heart-like  body.  The  abdomen  is 
composed  of  six  segments,  each  of  which  has  a  pair  of  stigmata,  but 
the  stigmata  of  the  first,  second,  third,  fourth,  and  fifth  segments  ap- 
pear to  be  in  one  broad  segment.  The  males  measure  i  mm.  in  length, 
the  females  1.5  mm.  These  lice  live  chiefly  in  the  pubic  hair  and  that 
of  the  perineum.  Rarely  they  are  found  in  the  axilla,  the  beard,  the 
eye-brows  and  even  upon  the  eye-lashes.  The  eggs  are  fixed  to  the 
bases  of  the  hairs  as  in  P.  capitis.  They  hatch  in  about  seven  days  and 
the  nymphs  grow  to  maturity  fifteen  days  later. 

The  bites  of  these  lice  are  very  irritating  and  cause  severe  itching  and 
the  eruption  of  pink  papules  that  sometimes  become  bluish  spots 
nearly  a  centimeter  in  diameter.  Such  spots  known  as  "  "taches 
ombrees"  are  frequent  in  typhoid  fever  when  lice  are  present. 

It  is  not  known  that  this  louse  can  harbor  spirochseta  or  any  patho- 
genic bacteria  or  protozoa. 


CHAPTER  XXI 
SLEEPING  SICKNESS 

TRYPANOSOMA  GAMBIEUSE  (DUTTON)  TRYPANOSOMA  RHODESIENSI 
(STEPHENS  AND  FANTHAM) 

SLEEPING  sickness,  African  lethargy,  Maladie  du  sommeil, 
Schlafkrankheit,  or  human  trypanosomiasis  is  a  specific,  infectious, 
endemic  disease  of  equatorial  Africa  characterized  by  fever,  lassi- 
tude, weakness,  wasting,  somnolence,  coma,  and  death.  The  first 
mention  of  the  disease  seems  to  have  been  made  by  Winterbottom.* 

Sir  Patrick  Mansonf  says  that  "For  upward  of  a  century  students 
of  tropical  pathology  have  puzzled  over  a  peculiar  striking  African 
disease,  somewhat  inaccurately  described  by  its  popular  name,  the 
sleeping  sickness.  Its  weirdness  and  dreadful  fatality  have  gained 
for  it  a  place  not  in  medical  literature  only,  but  also  in  general 
literature.  The  mystery  of  its  origin,  its  slow  but  sure  advance, 
the  prolonged  life  in  death  that  so  often  characterizes  its  terminal 
phases,  and  its  inevitable  issue,  have  appealed  to  the  imagination 
of  the  novelist,  who  more  than  once  has  brought  it  on  his  mimic 
stage,  draping  it,  perhaps,  as  the  fitting  nemesis  of  evil-doing.  The 
leading  features  of  the  strange  sickness  are  such  as  might  be  pro- 
duced by  a  chronic  meningo-encephalitis.  Slow  irregular  febrile 
disturbance,  headache,  lassitude,  deepening  into  profound  physical 
and  mental  lethargy,  muscular  tremor,  spasm,  paresis,  sopor,  ulti- 
mately wasting,  bed-sores,  and  death  by  epileptiform  seizure,  or  by 
exhaustion,  or  by  some  mtercurrent  infection. 

"In  every  case  the  lymphatic  glands,  especially  the  cervical, 
are  enlarged,  though  it  be  but  slightly.  In  many  cases  pruritus  is 
marked.  In  all,  lethargy  is  the  dominating  feature. 

"In  some  respects  this  disease,  which  runs  its  course  in  from 
three  months  to  three  years  from  the  oncoming  of  the  decided  symp- 
toms, resembles  the  general  paralysis  of  the  insane.  It  differs  from 
this,  however,  in  the  absence,  as  a  rule,  of  the  peculiar  psychic 
phenomenon  of  that  disease.  There  are  exceptions,  but  generally, 
though  the  mental  faculties  in  sleeping  sickness  are  dull  and  slow 
acting,  the  patient  has  no  mania,  no  delusions,  no  optimism.  So  far 
is  the  last  from  being  the  case,  that  he  is  painfully  aware  of  his  con- 
dition and  of  the  miserable  fate  that  is  in  store  for  him;  and  he  looks 
as  if  he  knew  it." 

*  "An  Account  of  Native  Africans  in  the  Neighborhood  of  Sierra  Leone,"  1803. 
t  "The  Lane  Lectures  for  1905,"  Chicago,  1905. 

506 


Specific  Organism  507 

Specific  Organism.— The  discovery  of  the  specific  organisms 
was  foreshadowed  byNepveu,*  who  recorded  the  existence  of  try- 
panosomes  in  the  blood  of  several  patients  coming  from  Algeria, 
by  Barron,f  and  by  Brault.J  In  1901  Forde  received  under  his 
care  at  the  hospital  in  Bathurst  (Gambia),  a  European,  the  captain 
of  a  steamer  on  the  River  Gambia,  who  had  navigated  the  river  for 
six  years,  and  who  had  suffered  several  attacks  of  fever  that  were 
looked  upon  as  malarial.  The  examination  of  his  blood  revealed 
the  presence  not  of  malarial  parasites,  but  of  small  worm-like  bodies, 
concerning  the  nature  of  which  Forde  was  undecided.  §  Later, 
Dutton,  in  conjunction  with  Forde,  examined  this  patient,  whose 
condition  had  become  more  serious,  and  recognized  that  the  worm- 
like  bodies  seen  by  Forde  were  trypanosomes.  Of  these  parasites 
he  has  written  an  excellent  description,  calling  them  Trypanosoma 


i 


V 


Fig.  206. — Trypanosoma  gambiense  (Todd). 

gambiense.||  The  patient  thus  studied  by  Forde  and  Dutton  died 
in  England  January  i,  1903.  In  1903  Dutton  and  Todd**  examined 
1000  persons  in  Gambia  and  found  similar  trypanosomes  in  the 
bloods  of  6  natives  and  i  quadroon.  In  the  same  year  Mansonft 
discovered  2  cases  of  trypanosomiasis  in  Europeans  that  had  be- 
come infected  upon  the  Congo.  Brumptf  J  also  observed  T.  gam- 
biense at  Bounba  at  the  junction  of  the  Ruby  and  the  Congo,  and 
Baker §§  observed  3  cases  at  Entebbe  in  Uganda. 

During  all  this  time  no  connection  was  suspected  between  these 

*  "Memoirs,  Soc.  de  Biol.  de  Paris,"  1891,  p.  49. 
"Transactions  of  the  Liverpool  Medical  Institute,"  Dec.  6,  1894. 
:  "Janus,"  July  to  August,  1898,  p.  41. 

§  "Trypanosomes  and  Trypanosomiasis,"  Laveran  and  Mesnil,  1907. 
||  See  Forde,  "Jour.  Trop.  Med.,"  Sept.  i,  1902;  Dutton,  Ibid.,  Dec.  i,  1902; 
Dutton,  "  Thompson- Yates  Laboratory  Reports,"  1902,  v,  4,  part  n,  p.  455. 

"*  "First  Report  of  the  Trypanosomiasis  Expedition  to  Senegambia,"  1902, 
Liverpool,  1903. 

ft  "Jour.  Trop.  Med.,"  Nov.  i,   1902,  and  March  16,   1903;  "Brit.  Med. 
Jour.,"  May  30,  1903. 

U  "Acad.  de  Med.,"  March  17,  1903. 
§§  "Brit.  Med.  Jour.,"  May  30,  1903. 


508 


Sleeping  Sickness 


micro-organisms  and  African  lethargy,  and  much  interest  was  being 
taken  in  a  coccus — 'the  hypnococcus — -that  was  being  studied  by 
Castellani  in  Uganda.  As  Castellani  was  prosecuting  the  investi- 
gation of  this  organism,  he  chanced  to  examine  the  cerebro-spinal 
fluid  of  several  negroes  in  Uganda  who  were  suffering  from  sleeping 
sickness,  and  in  it  found  trypanosomes.  Even  then,  though  Cas- 
tellani* realized  that  these  organisms  were  connected  with  sleeping 
sickness,  he  did  not  identify  them  in  his  mind  with  the  Trypano- 


Fig.  207. — Various  species  of  trypanosomes:  i,  Trypanosoma  lewisi  of  the  rat; 
2,  Trypanosoma  lewisi,  multiplication  rosette;  3,  Trypanosoma  lewisi,  small  form 
resulting  from  the  disintegrated  of  a  rosette;  4,  Trypanosoma  brucei  of  nagana; 
5,  Trypanosoma  equinum  of  caderas;  6,  Trypanosoma  gambiense  of  sleeping  sick- 
ness; 7,  Trypanosoma  gambiense,  undergoing  division;  8,  Trypanosoma  theileri, 
a  harmless  trypanosome  of  cattle;  9,  Trypanosoma  transvaliense,  a  variation  of 
T.  theileri;  10,  Trypanosoma  avium,  a  bird  trypanosome;  n,  Trypanosoma 
damonia  of  a  tortoise;  12,  Trypanosoma  solea  of  the  flat  fish;  13,  Trypanosoma 
grannlosum  of  the  eel;  14,  Trypanosoma  rajce  of  the  skate;  15,  Trypanosoma  rota- 
torium  of  frogs;  16,  Cryptobiaborreli  of  the  red-eye  (a  fish).  (From  Laveran  and 
MesniL) 

*  Ibid.,  May  23,  1903;  June  20,  1903. 


Morphology  509 

soma  gambiense  discovered  in  the  blopd  by  Forde  and  Button,  and 
described  the  newly  discovered  organism  as  Trypanosoma  ugan- 
dense.  Kruse,*  thinking  to  honor  the  discoverer,  called  it  Try- 
panosoma castellani.  Bruce  and  Nabarrof  found  the  new  try- 
panosome  in  each  of  38  cases  of  sleeping  sickness  in  the  cerebro- 
spinal  fluid,  and  12  out  of  13  times  in  the  blood.  These  observers 
also  found  that  23  out  of  28  natives  from  parts  of  Uganda  where 
sleeping  sickness  is  endemic  had  trypanosomes  in  their  blood,  while 
in  117  natives  from  uninfected  areas  the  blood  examination  was 
negative  in  every  case.  They  also  declared  that,  contrary  to  what 
had  been  stated,  there  were  no  appreciable  morphologic  differences 
between  Trypanosoma  gambiense  and  Trypanosoma  ugandense. 
Dutton,  Todd,  and  Christy!  arrived  at  the  same  conclusion.  The 
matter  was  finally  settled  by  Thomas  and  Linton§  and  Laveran,|| 
who,  by  means  of  animal  experiments,  determined  not  only  the 
complete  identity  of  the  organisms,  but  their  uniform  virulence. 

Early  in  1910  J.  W.  W.  Stephens**  studied  the  blood  of  a  rat  in- 
oculated with  blood  from  a  patient  suffering  from  sleeping  sickness, 
with  which  he  had  become  infected  in  North  Eastern  Rhodesia, 
and  observed  certain  definite  morphological  differences  between 
trypanosomes  in  it,  and  Trypanosoma  gambiense.  Later  he  and 
Fanthamft  studied  this  organism  with  great  care  and  came  to  the 
conclusion  that  it  was  a  new  and  separate  species,  and  gave  it  the 
name  Trypanosoma  rhodesiense.  In  this  they  received  the  support 
of  Mesnil.tt 

Morphology. — Trypanosoma  gambiense  is  a  long,  slender, 
spindle-shaped,  flagellate  micro-organism  that  measures  17  to  28  M 
in  length  and  1.4  to  2  M  in  breadth.  From  the  anterior  end  (that 
which  moves  forward  as  the  organism  swims)  a  whip-like  flagellum 
projects  about  half  the  length  of  the  organism.  The  terminal 
third  of  the  flagellum  is  free  in  most  cases.  The  proximal  two- 
thirds  are  connected  with  a  band  of  the  body  substance,  which  is 
continued  like  a  ruffle  along  one  side  of  the  organism  to  within  a 
short  distance  of  its  blunt  posterior  end,  where  the  flagellum  abruptly 
ends  at  the  blepharoplast.  This  thin  ruffle  is  known  as  the  un- 
dulating membrane.  By  means  of  the  flagellum  and  the  undulat- 
ing membrane  the  organism  swims  rapidly  with  a  wriggling  and 
rotary  movement  that  gives  it  the  name  Trypanosome,  which  means 
"  boring  body." 

*  "Gesell.  f.  natur.  Heilkunde,"  1903. 
t  "Brit.  Med.  Jour.,"  Nov.  21,  1903. 

j  Ibid.,  Jan.   23,  1904,  also  "  Thompson- Yates  and  Johnson  Lab.  Reports," 
1905,  v,  6,  part  i,  pp.  1-45. 

§  "Lancet,"  May  14,  1904,  pp.  1337-1340. 
"  Compt.-rendu  de  1'Acad.  des  Sciences,"  1906,  v,  142,  p.  1056. 
"British  Medical  Journal,"  1912,  n,  1182. 

ft  "Proceedings  of  the  Royal  Society,"  1910,  LXXXIII,  28,31;  i9i2,LXXXV,  223; 
"Bulletin  of  the  Sleeping-sickness  Bureau,"  1911-1912,  Nos.  33,  38. 
tt  "Brit.  Med.  Jour.,"  1912,  n,  1185. 


510  Sleeping  Sickness 

The  protoplasm  is  granular  and  often  contains  chromatin  dots 
that  are  remarkable  for  their  size  and  number.  There  is  a  distinct 
nucleus  of  ovoid  form  that  is  always  well  in  advance  of  the 
centrosome  or  blepharoplast,  and  not  infrequently  is  near  the  center 
of  the  organism.  There  is  also  a  centrosome  or  blepharoplast, 
which  appears  as  a  distinct,  deeply  staining  dot  near  the  posterior 
blunt  end  and  from  which  the  flagellum  appears  to  arise.  Near 
this  a  vacuole  is  sometimes  situated. 

Trypanosoma  rhodesiense  differs  from  Trypanosoma  gambiense  in 
that  the  nucleus  is  never  near  the  center,  rarely  far  in  advance  of  the 
blepharoplast,  and  not  infrequently  is  posterior  to  the  blepharoplast. 

Staining. — The  organisms  are  best  observed  when  stained  with 
one  of  the  polychrome  methylene-blue  combinations — Leishman's, 
Wright's,  Jenner's,  Romanowsky's,  Marino's.  To  stain  them  a 
spread  of  the  blood  or  cerebro-spinal  fluid  is  made  and  treated  pre- 
cisely as  though  staining  the  blood  for  the  differential  leukocyte 
count  or  for  the  malarial  parasite. 

Cultivation. — Trypanosoma  lewisi  of  the  rat  and  Trypanosoma 
brucei  of  "nagana"  or  ''tsetse-fly"  disease  of  Africa  have  been  culti- 
vated by  Novy  and  McNeal*  in  mixtures  composed  of  ordinary 
culture  agar-agar  and  defibrinated  rabbit-blood,  combined  as 
necessary,  1:1,  2:1,  1:2,  or  2:3,  etc.  The  actual  culture  was  made 
chiefly  in  the  water  of  condensation  collected  at  the  bottom  of 
obliquely  congealed  media. 

Laveran  and  Mesnil  found  that  when  blood  containing  Try- 
panosoma gambiense  was  mixed  with  salt  solution  or  horse-serum, 
the  trypanosomes  remain  alive  for  five  or  six  days  at  the  temperature 
of  the  laboratory.  They  live  much  longer  in  tubes  of  rabbit's 
blood  and  agar,  sometimes  as  long  as  nineteen  days,  and  during  this 
time  many  dividing  forms  but  no  rosettes  were  observed.  But 
subcultures  failed,  and  eventually  the  original  culture  died  out. 

Bayonf  has  found  it  easy  to  cultivate  Trypanosoma  rhodesiense 
in  Clegg's  ameba-agar  (q.v.)  and  in  blood  agar-agar  containing 
dextrose.  The  organisms  thus  cultivated  retain  their  virulence  for 
rats  for  a  long  time. 

Reproduction. — Multiplication  takes  place  by  binary  division, 
the  line  of  cleavage  being  longitudinal  and  beginning  at  the  posterior 
end.  The  centrosome  and  nucleus  divide,  then  the  flagellum  and 
undulating  membrane  divide  longitudinally,  and  finally  the  proto- 
plasm divides,  the  two  organisms  hanging  together  for  some  time  by 
the  undivided  tip  of  the  flagellum. 

In  addition  to  this  simple  longitudinal  fission,  the  trypanosomes 
seem  to  possess  a  sexual  mode  of  reproduction.  When  the  well- 
stained  organisms  are  carefully  studied,  it  is  possible  to  divide  them 

*  "Contributions  to  Medical  Research  dedicated  to  Victor  Clarence  Vaughan," 
Ann  Arbor,  Michigan,  1903,  p.  549;  "Journal  of  Infectious  Diseases,"  1904, 1,  p.  i. 
t  "Proc.  Royal  Society,  Series  B,"  1912,  LXXXV,  482. 


Transmission  511 

into  three  groups — those  that  are  peculiarly  slender,  those  that  are 
peculiarly  broad,  and  those  of  ordinary  breadth.  The  fact  that 
conjugation  takes  place  between  the  first  two  has  led  to  the  opinion 
that  they  represent  the  male  and  female  gametocytes  respectively, 
while  the  others  are  asexual.  All  forms  multiply  by  fission,  and 
conjugation  between  the  gametes  is  observed  to  take  place  only  in 
the  body  of  the  invertebrate  host.  It  has  not  yet  been  accurately 
followed  in  the  case  of  Trypanosoma  gambiense,  but  there  is  no 
reason  to  think  that  the  organism  differs  in  its  method  of  reproduc- 
tion from  Trypanosoma  lewisi.  Prowazek  found  that  when  rat 
blood  containing  the  latter  organism  was  taken  into  the  stomach 
of  the  rat  louse,  Hematopinus  spinulosus,  the  male  trypanosome 
enters  the  female  near  the  micronucleus  and  the  various  parts  of 
the  two  individuals  become  fused.  A  non-flagellate  ookinete  re- 
sults, and,  after  passing  through  a  spindle-shaped  gregarine-like 
stage,  can  develop  into  an  immature  trypanosome-like  form  in  the 
cells  of  the  intestinal  epithelium,  after  which  the  parasite  is  thought 
to  enter  the  general  body  cavity,  and,  migrating  to  the  pharynx, 
enter  the  proboscis,  through  which  it  is  transmitted  to  a  fresh 
host. 

Another  form  of  multiplication  consists  in  the  "shedding"  of 
infective  granules.  This  has  been  studied  by  Ranken.*  The  organ- 
isms from  which  this  is  about  to  take  place  are  observed  to  contain 
three  or  four,  sometimes  five  or  six  granules  of  small  size,  highly 
refractile  and  spherical  in  shape.  They  are  distinctly  within  the 
protoplasm  of  the  trypanosome  and  swing  backward  and  forward 
as  it  makes  its  lashing  movements.  When  these  are  closely  watched 
a  time  comes  when  one  of  the  granules  shoots  out.  At  first  the 
granule  is  carried  about  by  whatever  currents  of  fluid  it  happens  to 
meet,  having  no  motility  of  its  own,  but  soon  a  dot  appears,  then  a 
flagellum,  and  provided  with  means  of  locomotion,  and  now 
having  a  pyriform  shape,  the  new  embryo  parasite  swims  away. 
Ranken  thinks  these  granular  forms  develop  in  the  internal  organs 
and  has  found  them  of  pyriform  shape  in  the  liver,  spleen, 
and  lungs. 

Transmission. — -It  is  well  known  that  the  disease  does  not  spread 
from  person  to  person.  In  the  days  when  African  negroes  were 
imported  into  America  as  slaves,  the  disease  often  reached  our 
shores,  and  though  freshly  arrived  negroes  and  those  in  the  country 
less  than  a  year  frequently  died  of  it,  there  was  no  spread  of  the 
affection  to  those  that  were  acclimated.  The  Europeans  that 
carried  the  disease  from  Africa  to  England  and  were  the  first  in 
whose  bloods  the  trypanosomes  were  found,  did  not  spread  it  among 
their  fellow  countrymen.  A  case  from  the  Congo  that  died  in  a 
hospital  in  Philadelphia  and  came  to  autopsy  at  the  hands  of  the 
author,  did  not  spread  the  disease  in  this  city. 

*  "Brit.  Med.  Jour.,"  1912,  n,  408. 


Sleeping  Sickness 


Yet  the  disease  is  infectious,  and  the  transfer  of  a  small  quantity 
of  the  parasite-containing  blood  to  appropriate  experiment  animals 
perfectly  reproduces  it. 

The  present  knowledge  of  the  mode  of  transmission  came  about 
through  the  knowledge  of  other  trypanosome  infections  that  had 
already  been  carefully  studied  and  understood.  In  speaking  of 
nagana,  or  tsetse-fly  disease,  Livingstone,  as  early  as  1857,  recognized 
that  the  flies  had  to  do  with  it.  For  years,  however,  the  supposition 
was  that  the  fly  was  poisonous  and  that  its  venom  was  responsible  for 
the  disease.  In  1875  Megnin  stated  that  the  tsetse-fly  carries  a 
virus,  and  does  not  inoculate  a  poison  of  its  own.  In  1879  Drysdale 
suggested  that  the  fly  might  be  an  intermediate  host  of  some  blood 
parasite,  or  the  means  of  conveying  some  infectious  poison.  In 
1884  Railliet  and  Nocard,  who  suspected  the  same  thing,  proved 


Fig.  208. — Glossina  palpalis.  A 
perfect  insect  just  escaped  from  the 
pupa  (Brumpt).  Showing  how  the 
wings  close  over  one  another  like  the 
blades  of  a  pair  of  scissors. 


Fig.    209. — Glossina  palpalis  before 
and  after  feeding  (Brumpt). 


that  inoculations  with  the  proboscis  of  the  tsetse-flies  were  harmless. 
The  exact  connection  between  the  flies  and  the  disease  was  worked 
out  by  Bruce,*  who  found,  first,  that  flies  fed  on  infected  animals, 
kept  in  captivity  for  several  days,  and  afterward  placed  upon  two 
dogs,  did  not  infect;  second,  that  flies  fed  on  a  sick  dog,  and  imme- 
diately afterward  on  a  healthy  dog,  conveyed  the  disease  to  the 
latter.  The  flies  were  infectious  for  twelve,  twenty-four,  and  even  for 
forty-eight  hours  after  having  fed  on  the  infected  animal.  It  was, 
therefore,  shown  that  the  flies  could  and  did  infect,  not  through 
something  of  which  they  were  constantly  possessed,  but  through 
something  taken  from  the  one  animal  and  put  into  the  other;  this,  of 
course,  proved  to  be  the  trypanosome.  Further,  it  was  shown  that 
where  there  v/ere  no  tsetse-flies,  there  never  was  nagana. 

*  "Preliminary  Report  on  the  Tsetse-fly  Disease  or  Nagana  in  Zululand, 
Ubomfeo,  Zululand,"  Decw  1895;  "Further  Report,"  etc.,  Ubombo,  May  29, 
1896;  London,  1897. 


Transmission  513 

So  soon  a?  African  lethargy  was  shown  to  be  a  form  of  trypano- 
somiasis,  the  question  arose,  Was  it  spread  by  tsetse-flies?  Sambon* 
and  Brumptf  both  suggested  it,  but  it  was  soon  discovered  that  the 
geographic  distribution  of  the  tsetse-fly,  Glossina  morsitans,  that 
distributes  nagana,  does  not  coincide  with  the  geographic  distribu- 
tion of  sleeping  sickness.  There  are,  however,  different  kinds  of 
tsetse-flies,  and  Bruce  and  Nabarrof  first  showed  that  it  was  not 
Glossina  morsitans,  but  a  different  tsetse-fly,  Glossina  palpalis,  that 
is  the  most  important  source  of  the  spread  of  human  trypano- 
somiasis.  They  submitted  a  black-faced  monkey  (Cercopithicus) 
to  the  bites  of  numerous  tsetse-flies  caught  in  Entebbe,  Uganda,  and 
found  trypanosomes  in  its  blood.  Bruce,  Nabarro,  and  Greig§ 
allowed  Glossina  palpalis  to  suck  the  blood  of  negroes  affected  with 
sleeping  sickness  and  afterward  to  bite  five  monkeys  (Cercopithicus). 
At  the  end  of  about  two  months  trypanosomes  appeared  in  the  blood 
of  these  monkeys.  They  also  made  maps  showing  the  geographic 
distribution  of  African  lethargy  and  of  Glossina  palpalis,  which 
were  found  perfectly  to  correspond. 

But  the  natural  history  of  sleeping  sickness  is  less  simple  than  these 
facts  make  it  appear.  Kinghorn  and  Yorke||  observed  that  in 
the  Luangwa  Valley  where  tsetse-flies  (Glossina  morsitans)  abound, 
there  is  much  game  but  few  domestic  animals.  This  led  them  to 
study  the  bloods  of  all  the  game  animals  in  an  attempt  to  discover 
how  many  harbored  trypanosomes  and  what  kind  they  were.  The 
results  are  interesting,  but  two  are  of  great  importance  in  the  present 
connection.  They  discovered  that  antelopes  harbored  Trypano- 
soma  rhodesiense,  and  that  it  could  be  transmitted  by  Glossina 
morsitans.  As  Trypanosoma  rhodesiense  is  the  more  virulent 
parasite,  and  as  the  antelope  regularly  harbors  it  and  the  widely 
distributed  Glossina  morsitans  distributes  it,  the  likelihood  of 
an  early  and  successful  outcome  of  the  campaign  against  sleeping 
sickness  becomes  improbable. 

The  flies  are  found  to  become  infective  in  from  eleven  to  twenty- 
five  days  after  consuming  infected  blood,  and  to  remain  so  as 
long  as  they  continue  to  live. 

Bruce,  Hamerton,  Bateman  and  Mackie,  the  members  of  the 
" Royal  Society  Sleeping-sickness  Commission"  for  1908-9**  have 
found  that  under  experimental  conditions  the  development  of  the 
parasites  takes  place  only  in  about  5  per  cent,  of  infected  flies. 
The  shortest  time  in  which  their  flies  became  infective  was  18  days, 
the  longest  53  days,  the  average  34  days.  An  infected  fly  was  kept 

*  "Jour.  Trop.  Med.,"  July  i,  1903. 
t  "C.  R.  Soc.  de  Biol.,"  Jan.  27,  1903. 

i" Reports  of  the   Sleeping   Sickness   Commission  of  the  Royal  Society," 
1903,  i,  ii,  ii. 

§  Ibid.,  1903,  No.  4,  vm,  3. 

"Brit.  Med.  Jour.,"  1912,  n,  1186. 

"  British  Medical  Journal,"  1910,  i,  1312. 

33 


514  Sleeping  Sickness 

alive  in  the  laboratory  for  75  days  and  remained  infective  all  that 
time.  Experiments  directed  toward  finding  out  how  long  the  flies 
might  remain  infective  in  nature  indicate  that  the  flies  may  be  able 
to  transmit  the  parasites  for  at  least  two  years. 

It  is,  of  course,  not  impossible  that  other  flies,  especially  other 
species  of  tsetse-flies,  may  act  as  distributing  hosts  of  the  trypano- 
somes,  but  there  is  no  doubt  about  the  chief  agents  being  Glossina 
palpalis  and  Glossina  morsitans.  With  increased  entomologic 
and  geographic  information  it  has  been  found  that  there  are  certain 
districts  where  these  flies  abound  -though  the  disease  is  unknown, 
but  that  only  shows  that  in  those  districts  the  flies  are  not  infected. 
Tsetse-flies  are  not,  as  was  formerly  supposed,  peculiar  to  Africa, 
but  have  been  found  in  Arabia,  where  African  lethargy  could  no 
doubt  spread  should  the  flies  become  infected  through  imported 
cases  of  the  disease.  The  inability  of  the  disease  to  spread  in 
England  and  America  depends  upon  the  absence  of  tsetse-flies  from 
those  countries. 

It  is  possible  for  the  disease  to  be  transmitted  from  human  being 
to  human  being  through  such  personal  contacts  as  may  afford  oppor- 
tunity for  interchange  of  blood.  Thus,  Koch  observed  that  in 
certain  parts  of  Africa  where  there  were  no  tsetse-flies  the  wives  of 
men  that  had  become  infected  in  tsetse-fly  countries  sometimes 
developed  the  disease,  probably  through  sexual  intercourse,  a 
probable  explanation  when  one  remembers  that  it  is  solely  or  chiefly 
by  such  means  that  a  trypanosome  disease  of  horses — Dourine 
or  Maladie  du  coit,  caused  by  Trypanosoma  equiperdum — is 
transmitted. 

Transmission  to  Lower  Animals. — Trypanosoma  gambiense  is 
infectious  for  monkeys  as  well  as  for  human  beings.  In  the  monkeys 
a  diseas.e  indistinguishable  from  the  sleeping  sickness  is  brought 
about.  It  is  also  infective  for  dogs,  cats,  guinea-pigs,  rabbits,  rats, 
mice,  marmots,  hedgehogs,  goats,  sheep,  cattle,  horses,  and  asses. 
The  lower  animals  are  not,  however,  so  far  as  is  known,  subject  to 
natural  infection. 

Trypanosoma  rhodesiense,  being  a  more  virulent  parasite  than  its 
close  relative,  probably  infects  a  greater  variety  of  animals.  Among 
these,  in  nature,  antelopes  seem  to  be  commonly  infected. 

Pathogenesis. — The  first  effect  of  human  trypanosomiasis  seems 
to  be  fever  of  an  irregular  and  atypical  type,  occurring  in  irregular 
paroxysms.  It  was  in  this  early  febrile  stage  of  the  disease  that 
Forde  and  Dutton  first  found  the  trypanosom.es  in  the  circulating 
blood.  The  number  of  organisms  in  the  peripheral  circulation  is, 
however,  usually  so  small  that  it  is  tedious  to  look  for  them.  The 
search  may  be  made  in  thick  smears  stained  by  any  blood  stain,  but 
it  is  better  to  proceed  by  washing  the  corpuscles  in  citrated  blood 
as  in -preparing  to  calculate  the  opsonic  index,  and  to  collect  the 
"leukocyte  cream"  for  staining  and  examination.  The  trypano- 


Transmission  to  Lower  Animals  515 

somes,  which  seem  to  have  much  the  same  specific  gravity  of  the 
leukocytes,  appear  in  greatest  numbers  where  the  leukocytes  collect. 
In  African  natives  the  trypanosomes  may  be  present  in  the  blood 
for  a  long  time  before  any  symptoms  are  discovered,  but  in  Europeans 
their  presence  is  soon  followed  by  fever.  As  the  infection  progresses, 
the  micro-organisms  increase  in  great  numbers  in  the  organs,  and 
almost  entirely  disappear  from  the  blood.  The  lymph  nodes  swell 
and  Winterbottom,  who  first  described  the  disease,  called  particular 
attention  to  the  enlargement  of  those  of  the  posterior  cervical 
triangle,  which  he  regarded  as  of  diagnostic  significance. 

When  the  blood  examination  fails  to  reveal  trypanosomes,  they 
may  frequently  be  found  by  puncturing  an  enlarged  lymph  node 
with  a  dry  needle  and  examining  the  drop  of  fluid  obtained. 

Wolbach  and  Binger*  found  that  the  trypanosomes  invade  the 
connective- tissue  structure  of  all  organs,  the  reticular  tissue  of  lymph 
nodes  and  spleen,  and  the  substance  of  the  brain.  The  lesions  are 
due  to  the  presence  of  the  flagellated  form  of  the  parasite  in  the 
tissues.  They  found  the  initial  cell  reaction  to  be  the  proliferation 
of  endothelial  cells.  They  believe  the  discovery  of  numerous 
intravascular  mitoses  of  endothelial  cells  in  the  lung,  liver,  spleen 
and  kidney  to  indicate  the  source  of  the  increase  of  the  large  mono- 
nuclear  leukocytes  of  the  blood  in  human  trypanosomiasis. 

Lymphocytosis  is  the  rule  in  trypanosomiasis  but  is  of  no  diag- 
nostic importance. 

As  the  invasion  of  the  body  continues,  the  trypanosomes  dis- 
appear in  large  measure  from  the  blood  to  multiply  in  the  organs. 
In  the  spleen,  in  particular,  the  parasites  assume  a  different  form: 
a  deep  band  makes  its  appearance  between  the  nucleus  and  the 
blepharoplast.  The  former  becomes  surrounded  by  a  large  vacuole, 
and  the  trypanosome  becomes  disintegrated  and  reduced  to  a 
nucleus,  which  represents  the  latent  form  of  the  organism.  The 
nucleus  later  divides  giving  rise  to  a  new  blepharoplast  from  which  a 
new  flagellum  arises,  an  undulating  membrane  later  forms,  and  the 
usual  appearance  of  a  trypanosome  again  develops.  When  perfected , 
this  new  trypanosome  enters  the  circulating  blood.  At  the  time  that 
the  first  indications  of  somnolence  appear,  the  parasites  are  present 
in  the  cerebro-spinal  fluid.  The  fluid  is  collected  by  the  technic 
given  in  the  chapter  upon  cerebro-spinal  meningitis.  To  find  the 
trypanosomes  in  the  fluid,  it  should  be  rapidly  centrifugalized  for  a 
few  minutes  and  the  whitish  sediment  collected,  and  examined  imme- 
diately, when  the  micro-organisms  may  be  studied  alive,  or  the  fluid 
may  be  spread  upon  slides  and  stained  according  to  the  technic 
for  blood  spreads,  when,  the  trypanosomes  being  killed,  fixed  and 
stained,  their  structure  can  be  studied  to  advantage.  In  studying 
the  morbid  anatomy  of  sleeping  sickness,  Mottf  came  to  the  coii- 

'"Jour.  Med.  Research,"  1912-1913,  xxvii,  83. 
t "  British  Medical  Journal,"  Dec.  16,  1899,  n. 


•5*5 


Sleeping  Sickness 


elusion  that  the  essential  lesion  is  an  extensive  meningo-encephalitis. 
To  the  naked  eye,  there  are  scarcely  any  lesions  in  sleeping  sickness, 
except  the  enlargement  of  the  lymph  nodes,  and  even  in  the  nervous 
system  when  one  looks  with  care,  there  is  but  little  to  be  seen.  The 


Fig.  210. — Photomicrograph  of  an  eosin-methylene-blue-stained  section;  1000 
diameters.  Shows  trypanosomes  about  a  small  vessel  of  the  cortex  of  the  brain 
(Wolbach  and  Binger,  in  "Jour,  of  Med.  Research"). 

histological  examination  of  the  nervous  tissues,  on  the  contrary,  shows 
that  in  both  the  brain  and  spinal  cord  there  is  proliferation  and  over- 
growth of  neuroglia  cells,  especially  those  connected  with  the  sub- 


felfe» 


Fig.  211. — Photomicrograph  of 
a  Giemsa-stained  section;  1000 
diameters,  showing  a  trypano- 
some  deep  in  the  cortex  of  the 
brain  (Wolbach  and  Binger,  in 
''Jour,  of  Med.  Research"). 


©    (© 


Fig.  2 1 2. — Trypanosoma  gambiense. 
Formation  of  the  latent  stage  and 
transformation  of  the  latent  stage  into 
a  trypanosome  (after  Guiart). 


arachnoid  space  and  the  perivascular  space,  with  accumulation  and 
probably  proliferation  of  lymphocytes  in  the  meshwork.  Wohlbach 
and  Binger  found  that  the  trypanosomes  actually  escape  from  the 
blood-vessels  and  make  their  way  into  the  nervous  tissue.  The 


Prophylaxis  517 

period  of  lethargy  seems  to  coincide  with  that  at  which  the  parasites 
are  invading  and  injuring  the  nervous  tissue. 

Prophylaxis. — Reasoning  from  knowledge  of  the  successful  cam- 
paigns that  have  waged  against  yellow  fever  and  paludism,  it  at 
first  appeared  as  though  the  prophylaxis  of  sleeping  sickness  ought 
to  be  based  partly  upon  measures  taken  to  prevent  the  infection 
of  men  by  tsetse-flies,  and  partly  upon  those  taken  to  prevent  the 
infection  of  the  flies  by  men. 

To  prevent  the  infection  of  men  by  the  flies  is  extremely  difficult 
where  naked  or  half-naked  savages  are  to  be  dealt  with.  For 
Europeans,  the  customary  dress,  the  avoidance  of  exposure  in  bath- 
ing, the  use  of  mosquito  guards, etc.,  are  to  be  recommended,  as  well 
as  the  erection  of  habitations  and  the  building  of  roads,  etc.,  as 
far  as  possible  from  the  fly  districts.  The  destruction  of  the  grass 
and  reeds  along  the  river  banks,  the  use  of  drainage,  and  the  intro- 
duction of  chickens,  to  pick  up  the  larvae  and  pupae,  have  been 
recommended. 

To  prevent  infection  of  the  flies  with  Trypanosoma  gambiense  is 
impossible  where,  as  in  some  sections  of  Africa,  50  per  cent,  of  the 
population  of  some  of  the  villages  already  harbor  the  parasites, 
and  still  more  impossible  when,  as  is  the  case  with  Trypanosoma 
rhodesiense,  the  wild  animals,  especially  antelopes  which  are  ex- 
tremely numerous,  continually  harbor  the  parasites  and  act  as 
reservoirs  from  which  the  flies  receive  a  continuous  supply. 

The  importance  of  undertaking  radical  measures  for  the  prevention 
of  the  disease  may  be  imagined  when  it  is  understood  that  in  the 
last  few  years  no  less  than  a  half-million  of  the  natives  of  the  infected 
districts  have  died  of  sleeping  sickness. 

TSETSE  FLIES 

The  Tsetse  flies  are  dipterous  insects  belonging  to  the  family  Glossininae, 
and  included  in  a  single  genus  Glossina.  With  one  exception,  G.  tachinoides, 
the  entire  family  lives  in  tropical  and  subtropical  Africa.  About  sixteen 
species  of  Glossina  are  now  described,  for  the  rough  and  ready  identification 
of  which  the  following  table  from  Brumpt  ("Precis  de  Parasitologie "  1910,  p. 
630)  will  be  found  useful.  For  those  who  desire  more  accurate  information, 
Austin's  "Handbook  of  the  Tsetse  Flies,"  the  "Sleeping  Sickness  Bulletin," 
and  Patton  and  Cragg's  "Text-book  of  Medical  Entomology"  will  prove  useful 
books  of  reference. 

Tsetse  flies  are  easily  recognized  by  their  fly-like  appearance,  by  their  hori- 
zontal proboscis,  slender  but  swollen  at  the  base,  and  by  their  habit  of  resting 
with  the  wings  crossed  like  the  blades  of  a  closed  pair  of  scissors. 

The  greater  number  of  the  flies  occupy  sections  of  country,  spoken  of  as 
"fly  belts"  or  "fly  districts,"  some  of  which  are  permanently  infected,  others 
temporarily  infected.  Such  "belts"  are  usually  deep  forests  along  the  banks  of 
streams  or  on  the  shores  of  lakes.  The  adult  flies  seem  to  love  the  shade,  though 
they  fly  from  it  into  the  hot  sun  to  seek  their  prey.  The  large  game  animals 
seem  to  be  the  natural  prey  of  the  flies,  though  a  number  of  them  bite  human 
beings,  and  one,  Glossina  palpalis,  seems  to  prefer  human  blood  to  all  others. 
The  flies  seem  to  attack  moving  animals  by  preference.  So  long  as  the  creature 
moves  they  pursue.  When  it  stands,  many  of  them  fly  away  to  the  shade  again. 

Both  males  and  females 'bite.     The  latter  distend  themselves  with  blood  until 


518  Sleeping  Sickness 

they  are  so  heavy  that  they  can  scarcely  fly  and  drop  off  to  the  ground.  Biting 
is  almost  entirely  confined  to  bright  sunny  weather.  On  dull  or  cloudy  days  the 
flies  remain  in  the  brush.  Exceptions  are  found  among  the  few  species  that  live 
in  arid  sections.  Such  may  bite  at  night.  Few  of  the  flies  fly  far  from  their 
native  haunts  where  they  seem  to  prefer  to  await  the  coming  of  their  prey,  rather 
than  to  make  excursions  after  it.  Clouds  of  the  flies  often  arise  at  the  same  time 
and  attack  the  animals  in  swarms. 

The  flies  are  larviparous  and  do  not  lay  eggs.  Copulation  of  the  sexes 
takes  place  but  once,  the  sperm  being  retained  in  a  spermatotheca.  The 
eggs  are  fertilized  as  they  descend  from  the  oviduct  to  the  uterus  where  they 
hatch  into  a  larva  on  the  fifth  day.  The  larva  grows  rapidly,  molts  three  times  and 
attains  its  full  size  by  the  tenth  day,  when  it  is  born.  The  larva  at  the  time  of 
birth  is  cylindrical  in  shape,  consists  of  thirteen  segments  and  measures  6-7  mm.  in 
length.  It  is  nearly  white  but  has  a  black  head  which  is  small  and  incon- 
spicuous. The  larvae  are  usually  deposited  on  the  sand  of  the  banks  of  streams 
or  lakes,  and  at  once  burrow  into  the  ground  to  a  depth  of  an  inch  or  so.  In 
a  half  hour  or  an  hour  the  larva  changes  to  a  pupa  in  which  state  it  continues 
for  about  a  month.  The  imago  or  fly  then  emerges.  The  average  duration  of 
life  of  the  imago  fly  is  about  three  months,  during  which  time  each  female  bears  an 
average  of  ten  new  larvae. 

Glossina  palpalis  is  commonly  infested  by  a  flagellate  called  Crithidia  grayi, 
that  seems  in  some  way  to  pass  from  fly  to  fly,  and  to  have  nothing  to  do  with  the 
bloods  upon  which  it  feeds.  It  is  to  be  regarded  as  a  parasite  of  the  fly,  and 
should  be  known  lest  it  be  confused  with  the  Trypanosoma  of  which  the  fly  is 
the  vector. 

TABLE  FOR  THE  IDENTIFICATION  OF  THE  COMMON 
TSETSE  FLIES 

Large  Species;  body  measuring  more  than  1 2  mm.  in  length. 

Pattern  on  thorax  faint;  four  very  distinct  black  spots.  .  .  G.  longipennis. 

Pattern  on  thorax  sharp  and  distinct,  no  black  spots G.  fusca. 

Small  species;  body  in  general  measuring  less  than  1 2  mm.  in  length. 
All  five  tarsal  joints  of  the  third  pair  of  legs  black. 

Colors  dark;  antennae  black;  last  two  tarsal  joints  of  the 

first  pair  of  legs  black G.  palpalis. 

All  of  the  tarsal  joints  of  the  first  pair  of  legs  yellow.  .  .   G.  bocagei. 
Very  small  species;  markings  like  those  of  G.  morsitans  on 

abdomen G.  tachinoides. 

Colors  dark;  antennae  yellow G.  pallicera. 

Only  the  last  two  tarsal  joints  of  the  third  pair  of  legs  black; 

all  the  others  yellow. 
The  fifth  tarsal  joint  of  the  first  and  second  pairs  of  legs  is 

yellow G.  pallidipes. 

The  last  two  joints  of  the  tarsi  of  the  first  and  second  pairs  of  legs  are  black. 
The  yellow  band  on  the  abdominal  segments  takes  up 

one-third  of  the  segment G.  morsitans. 

The  yellow  band  on  the  abdominal  segments,  takes  up 

one-sixth  of  the  segment G.  longipalpis 

AMERICAN  TRYPANOSOMIASIS 
SCHIZOTRYPANUM    CRUZI    (CHAGAS) 

No  sleeping  sickness  has  thus  far  been  found  to  occur  upon  either 
of  the  American  continents,  though  human  trypanosomiasis  in 
another  form  has  been  observed  in  Brazil  where  it  has  been  studied 
by  Chagas.* 

*  "Archives  furschiffsu.  tropen  Hygiene,"  1909,  Heft  4;  abstract  "Centralbl. 
f.  Bacteriologie  etc.  Ref.,"  1909, XLIV,  639;  "Bull.  delTnst.  Pasteur,"  1910,  vra, 
373- 


American  Trypanosomiasis 


The  disease,  which  in  Minas  Gaeras  often  attacks  the  entire  popu- 
lation, chiefly  affects  the  children  and  goes  by  the  local  name  of 


Fig.  213.— i.  Glossma  paipalis  rf1  X  6.      2.  Glossina  morsitans,  9X6. 
(Patton  and  Cragg). 

Opilacao.  In  childhood  it  usually  assumes  the  form  of  an  acute 
malady  characterized  by  an  incubation  period  of  ten  days,  and  by 
high  continued  fever,  pufl&ness  of  the  face,  enlargement  of  the 


520  Sleeping  Sickness 

thyroid  gland,   of  the  lymph  nodes  and  spleen.     In  some  cases 
meningitis 'occurs.     It  is  extremely  fatal. 

In  adults  it  is  apt  to  take  a  more  chronic  course  in  which  the 
chief  symptoms  are  enlargement  of  the  thyroid  gland,  and  a  myx- 
edematous  condition  of  the  skin.  The  lymph  nodes  usually  en- 
large. If  the  adrenal  glands  become  affected,  symptoms  resembling 
Addison's  disease  make  their  appearance.  If  the  heart  muscle  be 
invaded  by  the  parasites,  its  power  is  diminished  and  the  pulse 
becomes  feeble  and  irregular.  If  the  nerve-cells  or  neuroglia  cells 
of  the  central  nervous  system  be  affected  through  parasitic  inva- 
sion, symptoms  occur  according  to  the  extent  and  localization 
of  the  disturbance.  There  is  always  irregular  fever  and  marked 
anemia. 

Chagas  found  a  trypanosome  in  the  peripheral  blood  of  patients 
suffering  from  Opilacao,  and  gave  it  the  name  Trypanosoma  cruzi. 
Later  studies  of  the  micro-parasite  have,  however,  shown  that  its 
method  of  reproduction  differs  §o  strikingly  from  that  of  the  trypano- 
somes,  that  it  was  necessary  to  make  a  generic  distinction  between 
the  two,  and  it  is  now  called  Schizotrypanum  cruzi. 

Morphology. — The  Schizotrypanum  is  present  in  the  peripheral 
circulation  only  during  the  febrile  stages  of  the  disease,  when  it 
may  be  found  by  the  usual  methods  of  staining  for  trypanosomes. 
It  is  a  long  slender  trypanosome-like  organism,  with  the  char- 
acteristic fusiform  shape,  with  a  nucleus,  a  large  blepharoplast,  a 
flagellum  and  an  undulating  membrane.  No  measurements  are 
given,  but  the  parasite  is  rather  small.  No  dividing  forms  are 
observed  in  the  circulating  blood.  The  trypanosomes  may  be  free, 
may  be  attached  to  the  erythrocytes  or  may  be  partly  or  entirely 
in  the  red  corpuscles.  They  show  sexual  dimorphism,  the  males 
being  long  and  slender,  the  females  shorter  and  stouter. 

Reproduction. — Gametogony  takes  place  in  the  lungs.  Such  of 
the  trypanosome  forms  as  are  caught  and  retained  there,  lose  the 
undulating  membranes,  the  two  ends  curve  toward  one  another 
forming  first  a  crescent,  them  unite  and  form  a  ring.  The  female 
parasites  shed  the  blepharoplasts,  and  in  both  male  and  female 
parasites  the  nucleus  breaks  up  into  eight  secondary  nuclei,  giving 
rise  to  eight  merozoits.  The  merozoits  derived  from  the  female 
parasites  have  a  single  nucleus,  those  derived  from  the  male  para- 
sites, a  nucleus  and  a  blepharoplast  connected  by  a  fine  thread  of 
chromatin.  The  merozoits  thus  formed  enter  into  erythrocytes 
where  they  eventually  develop  into  the  trypanosome  forms.  Hence 
is  explained  the  peculiar  relation  of  the  trypanosomes  to  the  eryth- 
rocytes mentioned  above. 

The  chief  multiplication  of  the  parasites,  however,  takes  place  in 
the  cells  of  the  voluntary  muscles,  the  heart  muscle,  the  central  nerv- 
ous system,  the  thyroid,  the  adrenal  glands  and  the  bone  marrow. 
In  these  situations,  according  to  Chagas,  the  parasites  take  on  a 


American  Trypanosomiasis  521 

rounded  form,  and  by  schizogony  give  rise  to  a  great  number  of 
daughter  parasites,  each  having  a  nucleus  and  a  blepharoplast. 
For  a  time  the  schizonts  are  quiescent,  then  develop  flagellae  and 
undulating  membranes.  The  infected  cells  are  destroyed.  Chagas 
thinks  that  gametes  are  formed  only  in  the  lungs. 

In  the  definitive  host,  the  Lamus  (or  Conorhinus)  megistis,  the 
sexual  conjugation  occurs  in  the  mid-gut.  The  blepharoplast 
approaches  and  seems  to  blend  with  the  nucleus,  the  undulating 
membrane  disappears  and  the  parasites  assume  a  spherical  form. 
Actual  conjugation  does  not  seem  to  have  been  observed.  Multi- 
plication takes  place  by  division  of  these  rounded  organisms,  the 
daughter  parasites  becoming  flagellated,  the  flagellum  originating 
from  the  blepharoplast.  Numerous  flagellated  trypanosome  and 
crithidia  forms  of  the  parasite  are  observed  in  the  hind-gut  of  the 
insect.  Chagas  observed  trypanosome  forms  in  the  body  cavity 
and  in  the  salivary  glands  by  the  insect,  and  it  is  probable  that  it  is 
through  these  that  the  infection  is  transmitted  when  the  insect 
bites  a  susceptible  animal,  though  Brumpt  thinks  the  infection  may 
take  place  through  the  feces  of  the  bug,  especially  when  these  are  in 
some  way  brought  to  the  conjunctiva. 

Transmission. — Chagas  was  able  to  show  that  a  large  bug, 
Lamus  (Conorhinus)  megistus,  common  in  the  neighborhood  in 
which  Opliacao  occurs,  is  the  principal  definitive  host  of  the  parasite. 
Both  males  and  females  of  this  flying  bug  are  vicious  biters  and  both 
live  upon  human  blood  as  well  as  upon  the  bloods  of  other  warm- 
blooded vertebrates.  The  bugs  are  common  in  the  thatch  and  in 
the  cracks  between  the  timbers  of  the  native  houses.  Whether 
other  species  of  Lamus  may  also  harbor  the  parasites  is  not  known. 
Brumpt*  found  that. Cimex  lectularius,  Cimex  boneti  and  Ornitho- 
dorus  moubata  could  also  act  as  definitive  hosts.  A  study  of 
Cimex  lectularius,  the  common  bed-bug,  as  a  definitive  host  of  the 
parasite,  was  made  by  Blacklockf  who  found  that  only  a  very 
occasional  bug  becomes  so  infected  as  to  be  able  to  effect  the 
transmission. 

Cultivation. — The  parasites  are  easily  cultivated  in  vitro  in  the 
medium  recommended  for  trypanosomes  by  Novy  and  McNeal. 
In  culture  the  organisms  resemble  those  found  in  the  bugs,  i.e.9 
round  and  crithidial  forms,  or  pear-shaped  rapidly  dividing  forms. 
More  than  two  subcultures  can  rarely  be  made  before  the  organisms 
die  out. 

Pathogenesis. — The  Schizotrypanum  is  pathogenic  for  certain 
monkeys  (Callithrix),  dogs,  rabbits  and  guinea-pigs.  Guinea-pigs 
usually  die  in  five  to  ten  days,  though  the  trypanosome  forms  are  not 
usually  found  in  the  peripheral  blood.  They  are,  however,  present 
in  larger  numbers  in  the  lungs.  Monkeys  live  longer.  Trypano- 

*"Centralbl.  f.  Bakt.  etc.  Ref.,"  LV,  No.  3,  p.  75. 
t  "British  Medical  Journal,"  1914, 1,  912. 


522 


Sleeping  Sickness 


some  forms  of  the  parasite  appear  in  the  blood  in  about  a  week, 
then  may  disappear.     The  animals  live  a  month  or  two. 
Diagnosis. — As  the  trypanosomes  are  present  in  the  circulating 


,: 


Fig.  214. — Schizotrypanum  cruzi  developing  in  the  tissues  of  the  guinea-pig. 
i.  Cross-section  of  a  striated  muscle  fiber  containing  Schizotrypanum  cruzi:  Note 
dividing  forms.  2.  Section  of  brain  showing  a  Schizotrypanum  cyst  within  a 
neuroglia  cell,  containing  chiefly  flagellated  forms.  3.  Section  through  the  supra- 
renal capsule,  fascicular  zone.  4.  Section  of  brain  showing  a  neuroglia  cell  filled 
with  round  forms  of  Schizotrypanum.  (From  Low,  in  Sleeping  Sickness  Bulletin, 
after  Vianna.) 


blood  of  human  beings  in  somewhat  small  numbers,  and  only  at 
certain  times,  it  is  unwise  to  rely  upon  them  as  a  means  of  making 


American  Trypanosomiasis  523 

the  diagnosis,  though  if  they  be  found  the  diagnosis  is  certain.  It 
is  usually  much  better  to  inoculate  i  or  2  cc.  of  the  blood  of  the 
suspected  case  into  a  guinea-pig  and  then  make  frequent  examina- 
tions of  its  blood.  Here,  again,  the  common  absence  of  trypanosome 
forms  from  the  blood  complicates  matters.  If  none  can  at  any  time 
be  found,  the  muscles  of  the  guinea-pig  must  be  examined  for  the 
dividing  forms  of  the  parasites,  which  are  usually  quite  numerous. 
Prophylaxis. — As  the  bugs  fly  it  is  somewhat  difficult  to  defend 
the  sleeping  patient  against  them,  so  long  as  he  lives  in  a  carelessly 
built  and  thatched  country  house.  Sulphur  fumigation  and  white- 
washing may  help.  Well-built  habitations  with  screened  windows 
and  the  use  of  mosquito  bars  should  constitute  the  best  defense. 

LAMUS  (CONORHINUS)  MEGISTIS  (BURN) 

Patton  and  Cragg*  describe  this  bug  as  follows :     "Dark  brown  to  black.     Pro- 
notum  broadly  expanded,  with  two  broad  raised  red  lines  extending  from  the 


Fig.  215. — Lamus  (Conorhinus)  megistus  (female),  the  insect  host  and  distributing 
agent  of  Schizotrypanum  cruzi  (Chagas).     X  2. 

middle  of  the  posterior  border,  and  a  red  spot  on  the  postero-lateral  angles  of  pro- 
notum.  At  the  anterior  border  of  the  pronotum  there  are  six  short  spines,  three 
on  each  side;  the  most  anterior  are  the  longest  and  project  on  each  side  of  the  eyes; 
two  are  situated  further  back,  one  on  each  side  of  the  middle  line  at  the  origin  of 
the  two  admedial  ridges;  the  third  spine  is  situated  on  a  ridge  at  the  junction  of 
the  middle  and  anterior  third  of  the  pronotum  just  above  the  first  pair  of  legs. 
Scutellum  dark  brown  with  two  short  red  lines  converging  toward  the  apex,  where 
they  meet;  apex  red,  turning  upward  and  bluntly  rounded  off.  Corium  and 
membrane  fuscous,  the  former  with  one  or  more  red  streaks.  Connexivum  with 
six  well-marked  bright  red  lines,  broader  in  the  male;  in  both  sexes  the  lines  ex- 
tend round  to  the  ventral  border.  In  the  male  the  last  segment,  except  for  a 
central  black  mark,  is  entirely  red.  Length  30  to  32  mm." 

The  L.  megistus  "is  almost  entirely  a  domestic  insect."     "The  adults  enter 

*  "A  Text-book  of  Medical  Entomology,"  1913,  p.  492. 


524  Sleeping  Sickness 

inhabited  houses  but  never  those  that  have  been  abandoned.  In  houses  which 
are  old  or  badly  kept  they  are  to  be  found  in  cracks  and  holes  in  the  walls,  where 
they  lay  their  eggs;  the  early  stages,  which  are  wingless,  crawl  out  of  their  resting 
places  in  the  walls  so  soon  as  the  lights  are  put  out  and  make  their  way  to  the  beds 
of  the  occupants  of  the  house.  The  adults  behave  in  the  same  manner,  but 
as  they  are  powerful  fliers,  they  can  reach  the  people  who  sleep  in  hammocks. 
The  bite  is  said  to  be  painless  and  to  leave  no  mark." 

"  The  eggs  of  L.  megistus  are  of  a  creamy  white  color  and  are  laid  in  batches  of 
from  eight  to  twelve,  and  as  many  as  forty-five  such  batches  may  be  laid.  Ac- 
cording to  Neiva  they  hatch  in  twenty-five  to  forty  days.  The  larva  is  of  a 
uniform  light  color  when  it  emerges,  becoming  darker  later;  it  takes  its  first 
feed  from  five  to  eight  days  after  emerging  from  the  egg,  and  the  second  from 
the  fifteenth  to  the  twentieth  day;  it  changes  its  skin  (first  nymphal  stage)  after 
about  forty-five  days.  The  second  molt  takes  place  during  the  second  or  third 
month,  and  the  third  during  the  fourth  or  sixth  month.  The  fourth  molt  occurs 
about  the  igoth  day  after  the  larva  has  hatched  out  from  the  egg;  this  stage 
lasts  at  least  forty-two  days.  Neiva  states  that  this  time  is  the  most  critical 
period  in  its  life,  and  that  large  numbers  of  them  die.  After  the  next  molt 
the  adult  stage  is  reached,  and  eight  days  later  they  are  ready  to  suck  blood; 
egg-laying  commences  about  the  fifty-fifth  day  after  the  first  feed.  One  female 
kept  under  observation  by  Neiva  for  about  three  and  a  half  months  laid  218 
eggs  in  thirty-eight  batches.  Under  favorable  conditions  of  food  supply  the 
cycle  from  egg  to  egg  is  completed  in  about  324  days." 

This  bug,  when  experimentally  infected  with  Schizotrypanum  cruzi,  transmitted 
the  infection  to  monkeys,  guinea-pigs,  rabbits  and  dogs.  Both  males  and  females 
bite  and  may  transmit  the  parasites. 


CHAPTER  XXII 
KALA-AZAR  (BLACK  SICKNESS) 

LEISHMANIA  DONOVANI  (LAVERAN  AND  MESNIL) 

"KALA-AZAR,"  " Dumdum  fever,"  "Febrile  tropical  spleno- 
megaly," "  Non-malarial  remittent  fever,"  is  a  peculiar,  fatal, 
infectious  disease  of  India,  Assam,  certain  parts  of  China,  the  Malay 
Archipelago,  North  Africa,  the  Soudan  and  Arabia,  caused  by  a 
protozoan  micro-organism  known  as  Leishmania  donovani,  and 
characterized  by  irregular  fever,  great  enlargement  of  the  spleen, 
anemia,  emaciation,  prostration,  not  infrequent  dysentery,  occa- 
sional ulcerations  of  the  skin  and  mucous  membranes,  and  sometimes 
cancrum  oris. 

Because  of  its  protean  manifestations  the  disease  has  been  given 
many  names,  and  has  been  confused  with  the  various  diseases  which 
its  symptoms  may  resemble.  It  was  not  until  1900  that  it  was 
finally  differentiated  from  malarial  fever  and  came  to  be  regarded 
as  a  distinct  entity. 

In  1900  Leishman*  noticed  in  the  spleen  of  a  soldier  returned 
from  India  and  suffering  from  "dumdum  fever" — a  fever  acquired 
at  Dumdum,  an  unhealthy  military  cantonment  not  far  from  Cal- 
cutta— certain  peculiar  bodies.  He  reserved  publishing  the  observa- 
tion until  1903,  so  that  it  appeared  almost  simultaneously  with  a 
paper  upon  the  same  subject  by  Donovan.")"  As  the  publications 
came  from  men  in  different  parts  of  the  world,  appeared  so  nearly 
at  the  same  time,  and  showed  that  they  had  independently  arrived 
at  the  same  discovery,  the  parasite  they  described  became  known 
as  the  Leishman- Donovan  body.  For  a  long  time  its  nature  was 
not  known  and  its  proper  classification  impossible,  but  after  it  had 
been  carefully  studied  by  Rogers,!  Ross,§  and  others,  and  its  de- 
velopmental forms  observed,  it  was  agreed  that  it  belonged  in  a  new 
genus  of  micro-organisms,  not  far  removed  from  the  trypanosomes, 
and  eventually  Ross,  and  then  Laveran  and  Mesnil,  honored  both 
of  its  discoverers  by  calling  it  Leishmania  donovani,  which  name  has 
been  generally  accepted. 

Morphology. — As  seen  in  a  drop  of  splenic  pulp  the  organism  is  a 
minute  round  or  oval  intracellular  body  measuring  2.5  by  3.5  p. 
When  properly  stained  with  polychrome  methylene  blue  (Wright's, 

*  "Brit.  Med.  Jour.,"  1903,  i,  1252. 
t  Ibid.,  1903,  n,  79. 

I" Quarterly  Jour.  Microscopical  Society,"  XLvm,  367;  "Brit.  Med.  Jour., 
1904,  i,  1249;  ii)  645;  "Proceedings  of  the  Royal  Society,"  LXXVII,  284. 
§  "Brit.  Med.  Jour.,"  1903,  n,  1401. 

525 


S26 


Kala-Azar 


Leishman's,  or  Jenner's  stains)  and  examined  under  a  high  magnifi- 
cation, it  is  found  that  the  protoplasm  takes  a  pinkish  color  and 
contains  two  well-defined  bright  red  bodies.  The  larger  of  these 
is  ovoid  and  lies  excentrically,  its  long  diameter  corresponding  to 


;*-f 


Fig.  216. — Evolution  of  the  parasite  of  kala-azar:  i  to  5.  Parasites  of  kala- 
azar.  i.  Isolated  parasites  of  different  forms  in  the  spleen  and  liver;  2,  division 
forms  from  liver  and  bone-marrow;  3,  mononuclear  spleen  cells  containing  the 
parasites;  4,  group  of  parasites;  5,  phagocytosis  of  a  parasite  by  a  poly  nuclear 
leukocyte.  6  to  15.  Parasites  from  cultures.  6,  First  changes  in  the  parasites. 
The  protoplasm  has  increased  in  bulk  and  the  nucleus  has  become  larger;  7, 
further  increase  in  size;  vacuolization  of  the  protoplasm;  8,  division  of  the  en- 
larged parasite;  9,  evolution  of  the  flagella;  10,  small  pyriform  parasite  showing 
flagellum;  n,  further  development  and  division  of  the  parasite;  12,  flagellated 
trypanosoma-like  form;  13,  14,  flagellated  forms  dividing  by  a  splitting  off  of  a 
portion  of  the  protoplasm;  15,  narrow  flagellated  parasites  which  have  arisen  by 
the  type  of  division  shown  in  13  and  14.  (From  Mense's  "Handbuch,"  after 
Leishman.) 

the  long  diameter  of  the  organism.  This  is  regarded  as  the  nucleus. 
The  second  body  is  smaller  and  of  bacillary  shape,  and  usually  lies 
with  its  long  diameter  transverse  to  the  nucleus.  This  is  looked  upon 
as  a  blepharoplast.  It  stains  more  intensely  than  the  nucleus. 


Cultivation  527 

In  addition  to  these  bodies  the  protoplasm  may  contain  one  or  two 
vacuoles. 

All  of  the  bodies  are  intracellular,  as  can  easily  be  determined 
by  examining  sections  of  tissue,  but  in  smears  of  splenic  pulp  the 
cells  are  broken  and  many  free  bodies  may  appear.  The  cells  in 
which  they  occur  are  lymphocytes,  endothelial  cells,  and  peculiar 
large  cells  whose  histogenesis  is  obscure.  They  are  rarely  to  be 
found  in  polymorphonuclear  leukocytes,  and  though  there  has 
been  much  discussion  upon  this  point,  probably  never  appear  in  the 
red  blood-corpuscles. 

The  bodies  divide  by  binary  and  multiple  fission,  without  rec- 
ognizable mitotic  changes.  When  multiple  fission  occurs,  the 
nucleus  divides  several  times  before  the  protoplasm  breaks  up.  The 
organism  is  not  motile  and  at  this  stage  has  no  flagella. 


Fig.  217. — Leishman-Donovan  bodies  from  the  spleen  of  a  case  of  kala-azar. 
X  about  1000.  (From  Beattie  and  Dickson's  "A  Text-book  of  General  Path- 
ology," by  kind  permission  of  Rebman,  Limited,  publishers.) 

Cultivation. — The  organism  was  first  cultivated  artificially 
by  Rogers  in  citrated  splenic  juice  at  17°  to  24°C.  It  can  also  be 
cultivated  in  the  blood-serum  agar  medium  used  by  Novy,  McNeal, 
and  Hall  for  trypanosomes,  and  in  the  N.  N.  N.  medium  of  Nicolle, 
which  has  the  following  composition: 

Water 900  cc. 

Salt  (NaCl) 6  gm. 

Agar-agar 16  gm. 

Dissolve,  distribute  in  tubes,  sterilize,  and  add  to  the  medium  in  each  tube 
after  liquefying  and  cooling  to  4o°-5o°C.,  one- third  of  its  volume  of  rabbit's 
blood  obtained  by  cardiac  puncture.  Slope  the  tubes  for  twelve  hours,  incubate 
at  37°C.  for  five  days  to  test  the  sterility  of  the  medium,  then  keep  at  the  ordinary 
temperature  of  the  laboratory,  sealed  to  prevent  evaporation. 

It  is  imperative  that  the  material  planted  be  sterile  so  far  as  bacteria 


Kala-Azar 


are  concerned.  Any  associated  growing  bacteria  quickly  destroy 
Leishmania  donovani. 

Under  conditions  of  cultivation  the  appearance  of  the  organism 
undergoes  a  complete  change.  It  enlarges,  the  nucleus  increases 
greatly  in  size,  and  a  pink  vacuole  appears  near  the  blepharoplast. 
In  the  course  of  twenty-four  to  forty-eight  hours  the  organism 
elongates,  the  blepharoplast  moves  to  one  end,  and  from  the  vacuole 
near  it  a  flagellum  is  developed,  and  the  organism  becomes  in  about 
ninety-six  hours  a  flagellate  protozoan  resembling  herpetomonas. 
It  now  measures  about  20  ^  in  length  and  3  to  4  /z  in  breadth,  its 
whip  or  flagellum  measuring  about  3  ju  additional.  It  is  also  motile, 
and,  like  the  trypanosomes,  swims  with  the  flagellum  anteriorly. 
There  is  no  undulating  membrane. 

This  may  be  regarded  as  the  perfect  or  adult  form  of  the  organ- 
ism. It  multiplies  by  a  peculiar  mode  of  division  first  observed  by 


Fig.  218. — Leishmania  donovani.     Flagellated  forms  obtained  in  pure  cultures 

(Leishman). 

Leishman.  Chromatin  granules,  a  larger  and  a  smaller,  appear 
in  the  protoplasm  in  pairs,  after  which,  through  unequal  longitudinal 
cleavage,  long,  slender,  almost  hair-like  individuals,  containing  one 
of  the  pairs  of  chromatin  granules,  are  separated.  These  were 
serpentine  at  first,  but  later,  as  they  grew  larger,  a  flagellum  was 
thrust  out  at  one  end. 

Distribution. — The  Leishman-Donovan  body  is  widely  distrib- 
uted throughout  the  body  of  the  patients  suffering  from  kala-azar. 
It  occurs  in  great  numbers  in  the  cells  of  the  spleen,  of  the  liver,  of 
the  bone-marrow,  and  in  the  ulcerations  of  the  mucous  membranes 
and  skin.  In  the  peripheral  blood  they  are  few  and  only  in  the  leuko- 
cytes. They  are  always  intracellular,  or  when  in  the  circulating 
blood  may  be  found  in  indefinite  albuminous  masses,  probably  de- 
stroyed cells.  The  number  in  a  cell  varies  up  to  several  hundred, 
such  great  aggregations  only  being  found  in  the  peculiar  large  cells 
of  the  spleen. 

Lesions. — The  splenomegaly  is  the  most  striking  lesion.  The 
change  by  which  the  enlargement  is  effected  is  not  specific.  The 


Transmission  529 

organ  is  not  essentially  changed  histologically,  but  seems  to  be  merely 
hyperplastic.  The  liver  is  enlarged,  but  here,  again,  specific  changes 
may  be  absent.  In  some  cases  a  pallor  of  the  centers  of  the  lobules 
may  depend  upon  numbers  of  parasite-containing  cells,  partly 
degenerated. 

The  yellow  bone-marrow  becomes  absorbed  and  red  tissue  takes 
its  place,  as  in  most  profound  anemias. 

Transmission. — Rogers'  observation,  that  the  round  bodies  grew 
into  flagellate  bodies  at  temperatures  much  below  that  of  the  human 
body,  led  Manson  to  conjecture  that  the  extrahuman  phase  of  the 
life  of  the  organism  took  place  at  similar  low  temperatures  in  the 
soil  or  in  water.  Patton*  found  that  a  number  of  cases  sometimes 
occurred  in  the  same  house,  while  neighboring  houses  were  free,  and 
thought  this  suggested  that  a  domestic  insect  might  be  the  distribut- 
ing host.  Later,  Patton  t  reported  a  very  thorough  study  of  insects 
in  relation  to  kala-azar,  in  which  after  a  long  series  of  experimental 
investigation,  he  came  to  the  conclusion  that  the  Indian  bed-bug, 
Cimex  rotundatus,  is  the  specific  invertebrate  host  of  Indian  kala- 
azar.  It  seems  that  in  order  that  the  parasites  shall  mature  in  the 
bed-bug,  and  undergo  those  changes  that  shall  result  in  the  insect's 
infectivity,  the  bug  must  receive  one  full  meal  of  the  infected  blood. 
If  a  second  meal  is  taken,  the  digestive  condition  in  the  bug's  alimen- 
tary canal  is  changed,  and  instead  of  continuing  to  develop,  the 
parasites  die  out.  When  the  conditions  are  all  favorable,  Patton 
found  that  the  flagellates  continued  to  multiply  actively  from  the 
fifth  to  the  eighth  day.  By  the  twelfth  day  practically  all  had 
reached  the  postflagellate  stage  and  were  only  found  in  the  stomach 
of  the  bed-bug.  These  results  convince  Patton  that  Cimex  rotund- 
atus is  the  definitive  host,  but  the  proof  is  lacking.  No  animal 
is  known  to  be  sufficiently  susceptible  to  Leishmania  donovani,  to 
acquire  anything  resembling  Kala-azar,  therefore  there  is  none 
that  the  bug  can  successfully  infect.  Human  experiment  with 
so  fatal  a  disease  being  out  of  the  question,  the  case  rests  at  this 
point.  Row{  has,  however,  shown  that  when  a  monkey,  Macacus 
sinicus,  is  inoculated  cutaneously  or  subcutaneously  with  a  three- 
weeks-old  culture  of  Xeishmania  donovani,  a  cutaneous  or  sub- 
cutaneous lesion  may  result.  This  may  facilitate  future  studies 
with  biting  insects. 

It  maybe,  however,  that  Patton  and  others  are  wrong  in  thinking 
that  the  flagellate  stage  at  which  the  parasites  arrive  in  the  bed-bug 
is  the  infective  stage,  and  have,  therefore,  gone  astray.  Bayon§ 
points  out  that  Leishmania  infantum  is  infective  for  dogs  and 
monkeys  in  the  rounded  or  oval  stages,  not  in '  the  elongate  or 
cultural  stages,  and  that  the  same  may  be  true  of  Leishmania  dono- 

*  "Scientific  Memoirs  of  the  Government  in  India,"  1907,  No.  27. 

"Brit.  Med.  Jour.,"  1912  n,  1194. 
t  "Brit.  Med.  Jour./'  1912,  n,  1196. 
§  "Brit.  Med.  Jour.,"  1912,  H,  1197. 

34 


530  Kala-Azar 

vani.  The  fleas,  which  are  the  vectors  of  infantile  kala-azar  among 
dogs,  show  only  the  rounded  and  oval  forms  of  the  parasites,  never 
the  flagellated  forms. 

Quite  recently  Patton  and  Donovan  have  been  successful  in  infect- 
ing puppies  with  Leishmania  donovani,  though  the  mature  dogs  seem 
never  to  be  infected,  the  examination  of  2000  street  dogs  in  Madras 
and  other  cities  failing  to  reveal  any  of  the  parasites  in  either  the 
liver  or  spleen.  Patton  inoculated  a  white  rat  with  3  cc.  of  an  emul- 
sion of  human  spleen  containing  the  oval  forms  of  Leishmania  dono- 
vani from  a  case  of  Indian  kala-azar,  and  fifteen  days  later  found  the 
spleen  several  times  the  normal  size  and  containing  large  numbers  of 
the  parasites. 

Diagnosis. — The  anemia  of  kala-azar  is  usually  not  profound. 
The  erythrocytes  number  about  3,000,000  in  ordinary  cases  and  the 
hemoglobin  is  correspondingly  diminished.  As  in  malaria,  there  is 
leukopenia,  but  it  is  usually  more  severe,  the  white  corpuscles  some- 
times being  as  few  as  600  to  650  per  cubic  millimeter  of  blood. 
The  enlargement  of  the  spleen  and  liver  suggest  malaria. 

The  only  certain  way  to  make  a  diagnosis,  except  in  those  rare 
cases  where  one  has  the  good  fortune  to  find  occasional  parasites 
in  the  leukocytes  of  the  circulating  blood,  is  by  hepatic  or  splenic 
puncture.  A  large  hypodermic  needle  should  be  used,  and  it  should 
be  carefully  sterilized.  It  should  by  preference  be  thrust  into  the 
liver  and  a  drop  of  fluid  secured  for  examination.  If  nothing  be 
found  it  may  later  be  necessary  to  puncture  the  spleen,  though  it 
is  dangerous  because  of  the  probability  of  subsequent  hemorrhage. 
If  decided  upon  as  a  justifiable  method  of  examination,  the  needle 
is  thrust  into  the  spleen,  and  a  bit  of  splenic  pulp  secured  by  firmly 
withdrawing  the  piston  of  the  attached  syringe. 

Before  making  such  a  puncture,  leukemia  should  be  excluded,  lest 
hemorrhage  occur. 

Treatment. — No  treatment  thus  far  tried  has  proved  successful. 
The  disease  is  usually  fatal,  and  in  certain  parts  of  India  whole 
towns  have  been  depopulated  by  it  and  the  fear  of  it. 


INFANTILE  KALA-AZAR 
LEISHMANIA  INFANTUM  (NICOLLE) 

Pianese*  found  infantile  kala-azar  in  Italy,  and  in  the  children 
suffering  from  it  he  was  able  to  find  the  Leishmania  infantum. 

Nicolle,f  while  in  Tunis,  observed  a  form  of  kala-azar  that  was 
peculiar  to  childhood  and  most  frequent  in  babies  of  about  two 
years  of  age.  Mesnil  has  identified  the  affection  with  a  disease 
known  as  "ponos"  in  Greece.  In  the  spleens  of  such  patients 

*  "Gaz.  Intern,  di  Medicin,"  1905,  vin,  8. 

t  "Ann.  de  1'Inst.  Pasteur,"  1909,  xxin,  361,  441. 


Tropical  Ulcer  531 

Nicolle  found  an  organism  that  was  not  distinguishable  either  by 
microscopic  examination  or  by  cultivation  from  Leishmania  dono- 
vani,  but,  finding  that  it  was  infectious  for  dogs,  he  came  to  the  con- 
clusion that  it  was  a  separate  species,  and  called  it  Leishmania 
infantum.  He  also  found  that  the  dogs  in  Tunis  frequently  suf- 
fered from  spontaneous  infection  from  this  parasite,  and  it  is  possible 
that  it  is  from  the  dogs  that  the  children  become  infected. 

Further  experiments  with  this  parasite  by  Nicolle  and  Comte 
have  shown  that  in  the  form  in  which  it  occurs  in  the  human  spleen 
it  is  capable  of  infecting  monkeys,  and  Novy  has  succeeded  in 
cultivating  the  organism  and  infecting  dogs  with  artificial  cul- 
tures containing  its  flagellate  forms. 

It  is  now  thought  by  many  that  infantile  kala-azar  and  Indian 
kala-azar  are  identical  diseases,  caused  by  identical  parasites.  In 
considering  the  probable  source  of  the  disease  Stitt*  says:  "It  has 
been  suggested  that  the  Mediterranean  basin  may  have  been  the 
original  focus  of  visceral  kala-azar  and  that  it  spread  thence  to  India 
by  way  of  Greece  and  the  Russian  Caucasus,  cases  having  been  re- 
ported from  districts  which  would  join  the  two  foci.  Just  as  chil- 
dren bear  the  brunt  of  malaria  in  old  malarial  districts  and  adults 
suffer  in  places  in  which  the  disease  has  been  more  recently  im- 
ported, so  by  analogy  we  may  consider  the  disease  as  of  more 
recent  introduction  in  India  ....  In  the  Mediterranean  basin 
there  is  a  natural  canine  Leishmaniasis  and  some  think  the  human 
form  may  be  contracted  from  the  dog  through  the  medium  of  the 


TROPICAL  ULCER 

LEISHMANIA  FURUNCULOSA  (FIRTH) 

In  India,  northern  Africa,  southern  Russia,  parts  of  China, 
the  West  Indies,  South  America,  and,  indeed,  most  tropical  countries, 
a  peculiar  intractable  chronic  ulceration  is  occasionally  observed, 
and  is  variously  known  as  Tropical  ulcer,  Oriental  sore,  Biscra  boil, 
Biscra  button,  Aleppo  boil,  Delhi  boil,  Bagdad  boil,  Jericho  boil, 
and  Buton  d'Orient.  It  has  long  been  known  as  a  specific  ulcerat- 
ing granuloma.  The  lesions,  which  begin  as  red  spots,  develop  into 
papules  which  become  covered  with  a  scaly  crust  which  separates, 
leaving  an  ulcer  upon  which  a  new  crust  develops.  The  lesion 
spreads  and  is  much  larger  when  the  crust  again  separates.  A 
purulent  discharge  is  given  off  in  moderate  quantities  and  the 
ulcer  becomes  deep  and  perpendicularly  excavated.  It  lasts  for 
months — sometimes  a  year  or  more — and  gradually  cicatrizes, 
forming  a  contracting  scar  that  is  quite  disfiguring  when  upon  the 
face.  The  lesions  may  be  single,  though  they  are  commonly  mul- 

*  Diagnosis  and  Treatment  of  Tropical  Diseases,  1914,  p.  75. 


532 


Kala-Azar 


Fig.  219. — "Jericho"  boil  (Masterman  in  "Journal  of  Hygiene"). 


Fig.  220. — Helcosoma  tropicum,  from  a  case  of  tropical  ulcer  ("Delhi  sore") 
smear  preparation  from  the  lesion  stained  with  Wright's  Romanowsky  blood- 
staining  fluid.  The  ring-like  bodies,  with  white  central  portions  and  containing 
a  larger  and  a  smaller  dark  mass,  are  the  micro-organisms.  The  dark  masses  in 
the  bodies  are  stained  a  lilac  color,  while  the  peripheral  portions  of  the  bodies,  in 
typical  instances,  are  stained  a  pale  robin's  egg  blue.  The  very  dark  masses  are 
nuclei  of  cells  of  the  lesion.  X  1500  approx.  (Wright).  (From  photograph  by 
Mr.  L.  S.  Brown.) 


Tropical  Ulcer 


533 


tiple,  as  many  as  twenty  sometimes  occurring  simultaneously.  It 
is  thought  that  recovery  is  followed  by  immunity. 

Organism. — In  1885  Cunningham*  described  a  protozoan  organ- 
ism found  in  the  tropical  ulcer,  the  observation  being  confirmed 
by  Firth,  f  who  called  the  bodies  Sporozoa  furunculosa.  Later, 
J.  H.  Wright t  studied  a  case  of  tropical  ulcer  and  found  bodies  pre- 
cisely like  the  Leishmania  donovani.  He  gave  it  the  name  Hel- 
cosoma  tropicum.  The  great  similarity  to  the  other  organisms  has 
led  more  recent  writers  to  identify  it  with  Leishmania,  but  as  it 
induces  a  local  and  not  a  general  infection  like  kala-azar,  it  is  now 
known  as  Leishmania  furunculosa. 

Cultivation. — The  organism  has  been  cultivated  by  Nicolle  and 


Fig.  221. — Oriental  sore  (Wellcome  Research  Laboratory) . 

Manceaux§  upon  the  same  media  and  in  the  same  manner  as 
Leishmania  donovani  and  Leishmania  infantum  with  which  these 
investigators  believe  it  to  be  identical.  Cultivation  was  also  success- 
fully achieved  by  Row. 

Pathogenesis. — The  virus  is  pathogenic  for  man,  monkeys  such 
as  Macacus  simius,  M.  cynomolgus,  M.  rhesus  and  M.  inuus,  and  for 
dogs.  The  same  effects  are  produced  whether  fresh  virus  from  a 
human  ulcer,  or  from  an  artificial  culture  be  employed.  In  dogs 
the  inoculations  produce  only  nodular  formations;  in  monkeys, 
nodules  like  those  in  human  beings  that  go  on  to  ulceration.  Intra- 
peritoneal  inoculations  usually  fail.  The  most  successful  inocula- 

'  "Scientific  Memoirs  by  Medical  Officers  of  the  Army  in  India,"  1884,  I. 
t  "British  Med.  Journal,"  Jan.  10,  1891,  p.  60. 
t  'Jour,  of  Med.  Research,"  1904,  x,  472. 
§  "Ann.  de  1'Inst.  Pasteur,"  1910,  xxrv,  683. 


534  Kala-Azar 

tions  are  made  beneath  the  skin  in  the  neighborhood  of  the  nose. 
One  successful  infection  with  the  parasite  usually  confers  immunity; 
unsuccessful  intraperitoneal  introduction  of  large  quantities  of 
culture  produce  no  immunity. 

Transmission. — The  disease  can  be  transmitted  by  inoculation 
from  human  being  to  human  being. 

The  usual  mode  of  transmission  is  not  known,  but  as  the  lesions 
usually  occur  where  the  body  surface  is  uncovered,  it  may  be  that 
flies  or  other  insects  act  as  vectors  of  the  parasites. 

Preventive  Inoculation. — Jackson*  is  authority  for  the  statement 
that  "the  Jews  of  Bagdad  recognized  that  tropical  ulcer  is  in- 
oculable  and  autoprotective  years  ago,  and  practised  vaccination 
of  their  children  upon  some  portion  of  the  body  covered  by  cloth- 
ing, in  order  that  their  faces  and  other  exposed  parts  of  the  body  be 
not  disfigured  by  the  ulcers  and  the  resultant  scars."  Nicollef 
sought  to  vaccinate  according  to  modern  methods  with  killed  and 
living  cultures  of  the  organism,  and  was  successful  when  he  first 
used  killed  culture,  then  after  a  year  a  live  culture,  and  then  three 
months  later  another  live  culture. 

Treatment. — Row{  has  endeavored  to  cure  already  existing 
lesions  by  vaccination,  and  has  met  with  what  seems  to  be  encour- 
aging success.  Cultures  of  the  organism  were  permitted  to  grow  for 
seven  days,  then  sterilized  with  glycerin.  Patients  can  bear  0.25 
cc.  at  a  dose,  there  is  little  febrile  reaction,  and  the  lesions  proceed 
to  heal  nicely. 

HISTOPLASMOSIS 

'    HISTOPLASMA  CAPSULATUM  (DARLING) 

In  1906  Darling,  §  working  at  the  Isthmus  of  Panama,  observed 
certain  cases  presenting  pyrexia,  anemia,  leukopenia,  splenomegaly, 
and  emaciation,  and  bearing  a  close  resemblance  to  kala-azar.  The 
disease  was  quite  chronic,  and  it  terminated  fatally.  When  ex- 
amined at  autopsy,  these  cases  showed  necrosis  with  cirrhosis  of 
the  liver,  splenomegaly,  pseudo-granulomata  of  the  lungs,  small 
and  large  intestines,  ulceration  of  the  intestines,  and  necrosis  of 
the  lymph  nodes  draining  the  injected  viscera.  The  lesions  seemed 
to  depend  upon  the  invasion  of  the  endothelial  cells  of  the  smaller 
lymph-  and  blood-vessels  by  enormous  numbers  of  a  small  en- 
capsulated micro-organism. 

The  organism  is  small,  round  or  oval  in  shape,  and  measures  i 
to  4  AI  in  diameter.  It  possesses  a  polymorphous,  chromatin  nucleus, 
basophilic  cytoplasm,  and  achromatic  spaces  all  enclosed  within  an 
achromatic  refractile  capsule. 

*  "Tropical  Medicine,"  Phila.,  P.  Blakiston's  Son  &  Co.,  1907,  p.  478. 
t"Annales  de  PInst.  Pasteur,"  Tunis,  1908. 
J"British  Medical  Journal,"  1912,  1,540. 

§  "Jour.  Amer.  Med.  Assoc.,"  1906,  XLVI,  1283;  "Archivof  Int.  Med.,"  1908, 
n,  107;  "Jour.  Exp.  Med.,"  1909,  xi,  515. 


Histoplasmosis 


535 


The  micro-organism  differs  from  the  Leishman-Donovan  body 
of  kala-azar  in  the  form  and  arrangement  of  its  chromatin  nucleus 
and  in  not  possessing  a  chromatin  rod.  The  distribution  of  the 
parasite  in  the  body  is  accomplished  by  the  invasion  of  the  con- 
tiguous endothelial  cells  of. the  smaller  blood-  and  lymph-vessels 
and  capillaries,  and  by  the  infection  of  distant  regions  by  the  dis- 
lodgment  of  infected  endothelial  cells  and  their  transportation 


Fig.  222. — Histoplasma  capsulatum.  Mononuclear  cells  from  the  lung  con- 
taining many  parasites  (Darling).  (Samuel  T.  Darling  in  "Journal  of  Experi- 
mental Medicine.") 

thither  by  the  blood-  and  lymph-stream.  Thus  the  skin,  intestinal, 
and  pulmonary  nodules  may  be  due  to  secondary  distribution  of 
the  parasite.  The  micro-organism  apparently  lives  for  a  con- 
siderable period  of  time  in  the  tissues,  because  in  the  older  areas  of 
necrosis  there  are  myriads  of  parasites  all  staining  well. 

The  mode  of  infection  and  portal  of  entry  are  unknown.  The 
parasite  has  neither  been  cultivated  nor  transmitted  by  inoculation. 

Believing  it  to  be  a  ne^  parasite,  Darling  has  suggested  that  it 
be  called  Histoplasma  capsulatum. 


CHAPTER  XXIII 
YELLOW  FEVER 

THE  bacteriology  of  yellow  fever  has  been  studied  by  Domingos 
Freire,*  Carmona  y  Valle,f  Sternberg,{  Havelburg,§  and  Sanarelli,|| 
but  all  of  their  work  has  been  shown  to  be  incorrect  by  the  interest- 
ing researches  and  very  conclusive  results  of  Finlay,**  Carter, ft 
Reed,  Carroll,  Lazear,  and  Agramonte,It  and  Reed  and  Carroll,  §§ 
which  have  proved  the  mosquito  to  be  the  definitive  host  of  an  in- 
visible micro-organism. 

Reed,  Carroll,  Lazear,  and  Agramonte,  ||||  constituting  a  Board 
of  Medical  Officers  "for  the  purpose  of  pursuing  scientific  investiga- 
tions with  reference  to  the  acute  infectious  diseases  prevalent  on  the 
island  of  Cuba,"  began  their  work  in  1900,  at  Havana,  by  a  careful 
investigation  of  the  relationship  of  Bacillus  icteroides  to  yellow 
fever.  By  a  most  careful  technic  they  withdrew  and  examined  the 
blood  from  the  veins  of  the  elbow  of  18  cases  of  yellow  fever,  mak- 
ing 48  separate  examinations  on  different  days  of  the  disease,  and 
preparing  115  bouillon  cultures  and  18  agar  plates,  every  examina- 
tion being  negative  so  far  as  Bacillus  icteroides  was  concerned. 
They  were  entirely  unable  to  confirm  the  findings  of  Wasdin  and 
Geddings,***  that  Bacillus  icteroides  was  present  in  blood  obtained 
from  the  ear  in  13  out  of  14  cases,  and  concluded  that  both  Sanarelli, 
and  Wasdin  and  Geddings  were  mistaken  in  their  deductions. 

In  lieu  of  the  remarkably  interesting  discoveries  of  Ronald  Ross 
concerning  the  relation  of  the  mosquito  to  malarial  infection,  the 
commissioners,  remembering  the  theory  of  Finlay,  ft  f  who  in  1881 

*  "Doctrine  microbienne  de  la  fievre  jaime  et  ses  inoculation  preventives," 
Rio  Janeiro,  1885. 

"Lecons  sur  1'etiologie  et  la  prophylaxie  de  la  fievre  jaune,"  Mexico,  1885. 
{"Report  on  the  Etiology  and  Prevention  of  Yellow  Fever,"  Washington, 
1891;  "Report  on  the  Prevention  of  Yellow  Fever  by  Inoculation,"  Washington, 
1888. 

§  "Ann.  de  PInst.  Pasteur,"  1897. 

||  "Brit.  Med.  Jour.,"  July  3,  1897;  "Ann.  del'Inst.  Pasteur,"  June,  Sept.,  and 
Oct.,  1897. 

!  "  Amer.  Jour.  Med.  Sci.,"  1891,  vol.  en,  p.  264;  "Ann.  de  la  Real  Academia," 
1881,  vol.  xviii,  pp.  147-169;  "Jour.  Amer.  Med.  Assoc.,"  vol.  xxxvin,  April  19, 
1902,  p.  993. 

"New  Orleans  Med.  Jour.,"  May,  1890. 

"Phila.  Med.  Jour.,"  Oct.  27,  190x5;  "Public  Health,"  vol.  xxvi,  1900,  p.  23. 
"Public  Health,"  1901,  vol.  xxvii,  p.  113. 
"Phila.  Med.  Jour.,"  Oct.  27,  1900. 

*  "  Report  of  the  Commission  of  Medical  Officers  Detailed  by  the  Authority 
of  the  President  to  Investigate  the  Cause  of  Yellow  Fever,"  Washington,  D.  C., 
1899.  " 

ttt  "  Annales  de  la  Real  Academia,"  1881,  vol.  xviii,  pp.  147-169. 

536 


Mosquitoes  and  Yellow  Fever 


537 


published  an  experimental  research  showing  that  mosquitoes  spread 
the  infection  of  yellow  fever,  and  the  interesting  and  valuable  ob- 
servations of  Carter*  upon  the  interval  between  infecting  and 
secondary  cases  of  yellow  fever,  turned  their  attention  to  the  mos- 
quito. Securing  mosquitoes  from  Finlay  and  continuing  the  work 


Fig.  223. — Stegomyia  fasciata   (Stegomyia  calopus}:   a,  female;  b,  male 
(after  Carroll). 

where  he  had  left  it,  they  found  that  when  mosquitoes  (Stegomyia 
fasciata  sen  calopus)  were  permitted  to  bite  patients  suffering  from 
yellow  fever,  after  an  interval  of  about  twelve  days  they  became  able 
to  impart  yellow  fever  through  their  bites.  This  infectious  char- 
acter, having  once  developed,  seemed  to  remain  throughout  the 
*  "New  Orleans  Med.  Jour.,"  May,  1900. 


538  Yellow  Fever 

subsequent  life  of  the  insect.  So  far  as  it  was  possible  to  deter- 
mine, only  one  species  of  mosquito,  Stegomyia  calopus,  served  as  a 
host  for  the  parasite  whose  cycles  of  development  in  the  mosquito 
and  in  man  must  explain  the  symptomatology  of  yellow  fever. 

In  order  to  establish  these  observations,  experimental  inocula- 
tions were  made  upon  human  beings  in  sufficient  number  to  prove 
their  accuracy.  Unfortunately,  Dr.  Lazear  lost  his  life  from  an 
attack  of  yellow  fever. 

Reed,  Carroll,  and  Agramonte*  came  to  the  following  conclusions: 

1.  The  mosquito  C.  fasciatus  [Stegomyia  calopus]  serves  as  the  intermediate 
host  of  the  yellow  fever  parasite. 

2.  Yellow  fever  is  transmitted  to  the  non-immune  individual  by  means  of  the 
bite  of  the  mosquito  that  has  previously  fed  on  the  blood  of  those  sick  with  the 
disease. 

3.  An  interval  of  about  twelve  days  or  more  after  contamination  appears  to  be 
necessary  before  the  mosquito  is  capable  of  conveying  the  infection. 

4.  The  bite  of  the  mosquito  at  an  earlier  period  after  contamination  does  not 
appear  to  confer  any  immunity  against  a  subsequent  attack. 

5.  Yellow  fever  can  be  experimentally  produced  by  the  subcutaneous  injection 
of  blood  taken  from  the  general  circulation  during  the  first  and  second  days  of  the 
disease. 

6.  An  attack  of  yellow  fever  produced  by  the  bite  of  a  mosquito  confers  im- 
munity against  the  subsequent  injection  of  the  blood  of  an  individual  suffering 
from  the  non-experimental  form  of  the  disease. 

7.  The  period  of  incubation  in  13  cases  of  experimental  yellow  fever  has  varied 
from  forty-one  hours  to  five  days  and  seventeen  hours. 

8.  Yellow  fever  is  not  conveyed  by  fomites,  and  hence  disinfection  of  articles 
of  clothing,  bedding,  or  merchandise,  supposedly  contaminated  by  contact  with 
those  sick  with  the  disease,  is  unnecessary. 

9.  A  house  may  be  said  to  be  infected  with  yellow  fever  only  when  there  are 
present  within  its  walls  contaminated  mosquitoes  capable  of  conveying  the  para- 
site of  this  disease. 

10.  The  spread  of  yellow  fever  can  be  most  effectually  controlled  by  measures 
directed  to  the  destruction  of  mosquitoes  and  the  protection  of  the  sick  against 
the  bites  of  these  insects. 

11.  While  the  mode  of  propagation  of  yellow  fever  has  now  been  definitely 
determined,  the  specific  cause  of  the  disease  remains  to  be  discovered.  v 

The  probability  that  Bacillus  icteroides  is  the  specific  cause 
and  is  transmitted  by  the  mosquito  is  so  slight  that  it  need  scarcely 
be  considered.  All  analogy  points  to  the  organism  being  an  animal 
parasite  similar  to  that  of  malarial  fever. 

With  this  positive  information  before  us,  the  prophylaxis  of 
yellow  fever  and  the  prevention  of  epidemics  of  the  disease  where 
sporadic  cases  occur  becomes  very  simple  and  may  be  expressed  in 
the  following  rules : 

1.  Whenever  yellow  fever  is  likely  to  occur,  the  breeding  places  of  mosquitoes 
should  be  destroyed  by  drainage.     Cisterns  and  other  necessary  collections  of 
standing  water  should  be  covered  or  secured. 

2.  Houses  should  have  the  windows  and  doors  screened  and  the  inhabitants 
should  use  bed  nets. 

3.  So  soon  as  a  case  of  fever  appears  it  should  be  removed  in  a  mosquito-proof 
ambulance  to  a  mosquito-proof  apartment  in  a  well-screened  hospital  ward  and 
kept  there  until  convalescent. 

*  Pan-American  Medical  Congress,  Havana,  Cuba,  Feb.  4-7,  1901;  Sanitary 
Department,  Cuba,  series  3,  1902. 


Prophylaxis  539 

4.  The  premises  where  such  a  case  has  occurred  should  be  fumigated  by  burn- 
ing pyrethrum  powder  (i  pound  per  1000  cubic  feet)  to  stun  the  mosquitoes, 
which  fall  to  the  floor  and  must  afterward  be  swept  up  and  destroyed. 

By  these  means  Major  W.  C.  Gorgas,*  without  expensive  disin- 
fection and  without  regard  for  fomites,  has  virtually  exterminated 
yellow  fever  from  Havana  and  from  the  Canal  Zone,  Panama,  where 
it  was  for  many  years  endemic. 

A  practical  point  connected  with  the  screens  is  given  in  the 
work  of  Rosenau,  Parker,  Francis,  and  Beyer,f  who  found  that 
to  be  effective  the  screens  must  have  20  strands  or  19  meshes  to 
the  inch.  If  coarser  than  this  the  stegomyia  mosquitoes  can  pass 
through. 

Reed  and  Carroll!  were  the  first  to  filter  the  blood  of  yellow 
fever  patients  and  prove  that  after  it  had  passed  through  a  Berke- 
feld  filter  that  kept  back  Staphylococcus  aureus,  it  still  remained 
infective  and  capable  of  producing  yellow  fever  in  non-immune 
human  beings. 

This  subject  was  further  investigated  by  Rosenau,  Parker,  Francis, 
and  Beyer,§  who  found  that  the  virus  was  even  smaller  than  the 
first  experiment  would  suggest,  as  it  not  only  passed  through  the 
Berkefeld  filter,  but  also  through  the  Pasteur-Chamberland  filter. 
The  filtrates  always  remained  sterile  when  added  to  culture-media. 

The  virus  has  not  been  artificially  cultivated. 

Prophylaxis. — Guiteras||  has  studied  the  effect  of  intentionally 
permitting  non-immunes  who  are  to  be  exposed  to  the  disease  to 
be  experimentally  infected  by  being  bitten  by  infected  mosquitoes, 
after  which  they  are  at  once  carefully  treated.  His  first  con- 
clusion was  that  "the  intentional  inoculation  gives  the  patient  a 
better  chance  of  recovery,"  but  the  danger  of  death  from  the  ex- 
perimental infection  was  later  shown  to  be  so  great  that  it  had  to 
be  abandoned. 

*  International  Sanitary  Congress  held  at  Havana,  Cuba,  Feb.  16,  1902: 
Sanitary  Department,  Havana,  series  4. 

t  Report  of  Working  Party  No.  2,  Yellow  Fever  Institute,  Bull.  14,  May,  1904. 

t"Am.  Med.,"  Feb.  22,  1902. 

§  "Bull.  No.  14,  U.  S.  Public  Health  and  Marine  Hospital  Service,"  Washing- 
ton, D.  C.,  May,  1904. 

U  "Revista  de  Medicina  Tropical,"  Havana,  Cuba,  1902. 


CHAPTER  XXIV 
TYPHUS  FEVER 

TYPHUS  fever,  also  known  as  jail-fever,  ship-fever,  army-fever, 
and  by  a  large  number  of  other  names,  of  which  about  a  hundred 
have  been  collected  by  Murchison,*  has  long  been  known,  but  was 
probably  not  recognized  as  a  definite  disease  before  1760,  when 
Gaul  tier  de  Sauvage  endeavored  to  give  it  individuality,  or  1769 
when  Cullum  of  Edinburgh  defined  it.  Its  eventual  separation 
from  typhoid  fever,  with  which  it  continued  to  be  confused,  was 
the  result  of  the  studies  of  Gerhard  "On  the  Typhus  Fever  which 
occurred  in  Philadelphia  in  the  Spring  and  Summer  of  1836,  Etc."f 
The  Germans  still  speak  of  typhus  abdominalis,  meaning  typhoid 
or  enteric  fever,  and  typhus  exanthematicus ,  meaning  the  typhus 
fever  of  the  present  day.  The  Spanish  and  Mexicans  call  it  tabardillo. 

The  disease  is  largely  a  disease  of  poverty,  filth  and  crowding,  and 
is  of  frequent  occurrence  both  in  sporadic  and  epidemic  form  where 
such  conditions  occur  permanently  or  temporarily.  Its  most 
common  epidemic  occurrence  is  therefore  among  the  slums,  in  jails, 
in  ships,  in  asylums,  in  hospitals  and  in  armies.  With  the  improved 
hygienic  conditions  of  the  present  time  its  occurrence  in  consider- 
able epidemics  is  much  diminished,  and  it  is  not  to  be  expected  in 
sanitary  dwellings,  among  cleanly  people  or  in  well-regulated 
institutions. 

It  is  undoubtedly  transmissible  and  therefore  infectious,  but 
it  early  became  clear  that  the  infection  was  not  air-borne  and  did 
not  readily  pass  from  individual  to  individual.  Further,  it  *seems 
clear  that  the  survival  of  an  attack  confers  immunity  against  future 
infection. 

Though  its  infective  and  micro-organismal  nature  is  clear,  the 
specific  micro-organism  has  not  yet  been  discovered.  This  is  not 
because  it  has  not  been  made  the  subject  of  much  investigation  in 
many  countries  by  capable  men,  but  rather  because  of  peculiar 
circumstances  that  make  the  discovery  difficult,  if  not  impossible. 

The  early  investigations  of  the  subject  were  confined  to  dem- 
onstrating the  truly  infectious  nature  of  a  disease  whose  transmissi- 
bility  was  so  uncertain  as  to  permit  the  escape  of  large  numbers  of 
those  exposed  to  it. 

In  1876  MoczutkowskiJ  inoculated  himself  with  the  blood  of  a 

*  "A  Treatise  on  the  Continued  Fevers  of  Great  Britain,"  3d  edition,  1884,  p. 
161. 

t  Araer.  Jour. of  the  Med.  Sciences,  1836,  xix,  p.  283;  1837,  xx,  p.  289. 
$"Allgemeine  Med.  Central  Zeitung,"  1900,  LXVIIT,  1055. 

540 


Transmission  541 

patient  suffering  from  typhus  fever,  and  developed  the  disease 
eighteen  days  later.  In  1907  Otero*  endeavored  to  induce  the  dis- 
ease in  human  beings  by  inoculation.  In  one  out  of  four  attempts 
he  was  successful. 

Experiments  with  a  not  infrequently  fatal  malady  made  upon 
human  beings  being  immoral  and  inexpedient,  it  became  necessary 
to  find  some  animal  susceptible  to  the  disease,  with  which  further 
experiments  could  be  prosecuted. 

In  1909  Nicolle  f  succeeded  in  producing  the  disease  in  a  chim- 
panzee by  inoculating  it  with  human  blood.  Later  {  he  was  able  to 
transmit  the  disease  from  the  chimpanzee,  and  still  later  from  human 
beings,  to  Macacus  sinicus  by  inoculating  with  human  blood.  In 
1909  Anderson  and  Goldberger§  were  successful  in  transmitting 
the  disease  to  monkeys,  by  inoculating  them  with  human  blood. 
Other  workers  corroborated  these  results,  and  thus  it  became  clear 
that  the  suspicion  that  the  disease  was  infectious  was  correct, 
and  that  the  infectious  agent  was  in  the  blood  with  which  it  could 
be  carried  over  to  new  men  and  animals  and  reproduce  the  disease. 
Later  Nicolle,  Couer  and  Conseil||  were  able  to  transmit  the  disease 
to  guinea-pigs. 

In  Mexico,  Gaveno  and  Girard**  were  able  to  carry  the  infection 
through  1 1  transplantations  from  guinea-pig  to  guinea-pig,  and  still 
find  it  infective  for  monkeys. 

Still,  however,  the  micro-organism  could  not  be  found.  Two 
additional  problems  therefore  became  important  for  solution. 
First,  what  was  the  nature  of  this  virus  that  could  not  be  found, 
second,  how  did  it  naturally  pass  from  patient  to  patient? 

In  October,  1910,  Nicolle,  Couer  and  Conseilff  instead  of  working 
with  artificially  defibrinated  blood,  permitted  the  blood  to  coagulate 
spontaneously,  then  passed  it  through  the  most  porous  kind  of  a 
Berkefeld  filter,  and  successfully  infected  one  out  of  two  monkeys 
injected  with  the  filtrate.  After  other  series  of  experiments,  these 
investigators  came  to  the  conclusion  that  the  serum  of  artificially 
defibrinated  blood,  when  filtered,  was  always  without  infective 
power,  and  that  of  spontaneously  coagulated  blood,  commonly 
so,  and  that  hence,  though  the  virus  of  the  disease  is  a  filterable  virus, 
it  consists  of  organisms  so  large  as  to  be  commonly  held  back  by  the 
coarsest  Berkefeld  filters.  It  may  be  too  small  to  be  visible  never- 
theless, at  least  to  such  methods  of  observation  as  are  now  in  vogue. 

In  regard  to  the  transmission  of  the  disease  the  investigators  had 
before  them  the  usual  exemption  of  physicians,  nurses,  attendants 

*"Mem.  pres.  a  1'Acad.  de  Med.  de  Mex.,"  1907. 

f  "Ann.  de  1'  Inst.  Pasteur,"  1910,  xxiv. 

J"Compt.-rendu  Acad.  d.  Sciences  de  Paris,"  1910. 

§  "Public  Health  Reports,"  1909,  xxiv,  p.  1941. 

|]  "Ann.  de  1'Inst.  Pasteur,"  1910,  xxv,  97. 
**"Publ.  de  1'Inst.  Bact.  Noc.  Mex.,"  1910,  Nov.  9. 
ft  "Ann.  de  1'Inst.  Pasteur,"  1911,  xxv,  97. 


542  Typhus  Fever 

and  others  who  cared  for  patients  suffering  from  the  disease,  as 
contrasted  with  its  persistent  spread  to  new  patients  at  the  foci  of 
infection.  They  also  had  the  recently  gained  knowledge  of  the  part 
played  by  insects  and  arthropods  in  the  transmission  of  malaria, 
relapsing  fever,  African  lethargy,  etc.,  the  whole  matter  being  of 
such  nature  as  to  make  the  conclusion  that  the  infection  was  trans- 
mitted by  an  insect  host,  a  justifiable  one. 

The  first  to  work  upon  this  problem  were  Nicolle,  Couer  and 
Conseil,*  the  selected  insects  being  pediculi.  They  permitted  lice 
to  feed  upon  the  blood  of  an  infected  monkey,  and  then  upon  a 
healthy  monkey.  The  healthy  monkey  contracted  typhus  fever. 
In  the  same  year,  and  working  independently,  Goldberger  and 
Andersonf  made  two  attempts  to  infect  healthy  monkeys  by  per- 
mitting lice  fed  upon  cases  of  typhus  fever  in  men,  to  bite  them. 
They  had  partial  success — the  monkeys  became  diseased  but  no 
immunity  tests  were  made  for  confirmation  of  the  nature  of  the 
disease. 

Ricketts  and  Wilder  J  working  in  Mexico  succeeded  in  transmitting 
typhus  fever  from  man  to  monkeys  by  means  of  lice — Pediculus 
vestimenti.  They  also  succeeded  in  transmitting  the  disease  to  a 
monkey  by  scarifying  its  skin  and  applying  the  abdominal  contents 
of  some  infected  lice,  so  that  it  was  proved  by  them  that  the  cause 
of  infection  was  in  the  lice.  Later  Nicolle  and  Conseil  §  also  suc- 
ceeded in  infecting  a  monkey  by  the  bites  of  infected  lice. 

Wilder  1 1  further  found  that  the  infectious  agent  passes  from  the 
infected  lice  to  a  second  generation  of  insects,  as  does  the  spiro- 
chaeta  of  relapsing  fever  to  subsequent  generations  of  ornithodorus 
ticks.  Wilder  failed  in  experiments  directed  toward  infecting 
monkeys  by  fleas  or  bed-bugs. 

In  the  experiments  recorded  by  Wilder,  the  transmission  of  typhus 
fever  to  monkeys,  by  lice,  was  successful  in  7  out  of  \o  attempts. 
It  required  17  lice  to  infect  a  monkey.  In  one  case  a  monkey 
seemed  to  be  immunized  by  being  bitten  by  very  young  lice. 

Goldberger  and  Anderson**  also  experimented  with  the  head 
louse  Pediculus  capitus  and  succeeded  in  showing  that  it  too  takes 
up  the  typhus  fever  virus  and  may  pass  it  on  from  human  being  to 
monkey,  and  hence  probably  from  man  to  man. 

A  description  of  the  lice  will  be  found  in  the  chapter  upon  "Re- 
lapsing Fever." 

*"Compt.-rendu  de  1'Acad.  des  Sciences  de  Paris,"  1909,  CXLIX,  486. 
f  "Public  Health  Reports,"  1910,  xxv. 
j  "Jour.  Amer.  Med.  Asso.,"  1910,  LIV,  1304. 

§  "Compt.-rendu.  de  1'Acad.  des  Sciences  de  Paris,"  1911,  CLIII,  1522. 
||  "Journal  of  Infectious  Diseases,"  1911,  ixi. 
**  "Public  Health  Reports,"  1912,  xxvu. 


CHAPTER  XXV 
PLAGUE 

BACILLUS  PESTIS  (YERSIN,  KITASATO) 

General  Characteristics. — A  minute,  pleomorphous,  diplococcoid  and  elongate, 
sometimes  branched,  non-motile,  non-flagellated,  non-sporogenous,  non-liquefy- 
ing, non-chromogenic,  aerobic,  pathogenic  organism,  easily  cultivated  artificially, 
and  susceptible  of  staining  by  ordinary  methods,  but  not  by  Gram's  method. 

Plague,  bubonic  plague,  pest,  black  plague,  "black  death," 
or  malignant  polyadenitis  is  an  acute  epidemic  infectious  febrile 
disease  of  an  intensely  fatal  nature,  characterized  by  inflammatory 
enlargement  and  softening  of  the  lymphatic  glands,  marked  pul- 
monary, cerebral  and  vascular  disturbance,  and  the  presence  of  the 
specific  bacillus  in  the  lymphatic  nodes  and  blood. 

The  history  of  plague  is  so  full  of  interest  that  many  references 
to  it  appear  in  popular  literature.  The  student  can  scarcely  find 
more  profitable  reading  than  the  "History  of  the  Plague  Year  in 
London,"  by  DeFoe,  and  readers  of  Boccacio  will  remember  that 
it  was  the  plague  epidemic  then  raging  in  Florence  that  led  to  the 
isolation  of  the  group  of  young  people  by  whom  the  stories  of 
the  Decameron  were  told. 

During  the  reign  of  the  Emperor  Justinian  the  plague  is  said 
to  have  carried  off  nearly  half  of  the  population  of  the  Roman  Em- 
pire. In  the  fourteenth  century  it  is  said  to  have  destroyed  nearly 
twenty-five  millions  of  the  population  of  Europe.  Epidemics  of 
less  severity  but  attended  with  great  mortality  appeared  in  the 
sixteenth,  seventeenth,  and  eighteenth  centuries.  In  1894  an 
epidemic  broke  out  in  the  western  Chinese  province  of  Yunnan 
and  reached  Canton  in  January,  1894,  thus  escaping  from  its  en- 
demic center  and  began  to  spread.  It  can  be  traced  from  Canton 
to  Hongkong.  In  1895  ^  appeared  also  in  Amoy,  Macao,  and 
Foochoo.  In  1896  it  had  reached  Bombay  and  reappeared  in  Hong- 
kong. In  1897  Bombay,  the  Madras  Presidency,  the  Punjab,  and 
Madras  were  visited.  In  1898  the  disease  spread  greatly  through- 
out India  and  into  Turkestan,  and  by  sea  went  to  Madagascar  and 
Mauritius.  In  1899  it  extended  still  more  widely  in  India  and 
China,  Japan  and  Formosa,  and  succeeded  in  disseminating  as 
widely  as  the  Hawaiian  Islands  and  New  Caledonia  on  the  east, 
Portugal,  Russia,  and  Austria  on  the  west,  and  Brazil  and  Para- 
guay on  the  south.  In  1900  it  had  spread  to  nearly  every  part  of 
the  world.  In  those  places  in  which  sanitary  measures  could  not  be 
carried  into  effect  the  people  died  in  great  numbers — thus  in  India 

543 


544  Plague 

in  1901  there  were  362,000  cases  and  278,000  deaths.  In  the  first 
six  months  of  the  epidemic  of  1907,  the  deaths  in  India  were  much 
more  numerous,  reaching  a  total  of  1,062,908.  Where  sanitary 
precautions  are  possible  and  co-operation  between  the  people  and 
the  authorities  can  be  brought  about,  as  in  New  York,  San  Fran- 
cisco, and  other  North  American  and  European  ports,  the  disease 
remains  confined  pretty  well  within  limits  and  does  not  spread. 
An  interesting  account  of  "The  Present  Pandemic  of  Plague"  by 
J.  M.  Eager,  was  published  in  1908  in  Washington,  D,  C.,  by  the 
U.  S.  Public  Health  and  Marine  Hospital  Service. 

Plague  is  an  extremely  fatal  affection,  whose  ravages  in  the 
hospital  at  Hongkong,  in  which  Yersin  made  his  original  observa- 
tions, carried  off  95  per  cent,  of  the  cases.  The  death-rate  varies  in 
different  epidemics  from  50  to  90  per  cent.  In  the  epidemic  at 


.  j 


Fig.  224. — Axillary  bubo.   (Reproduced  from  Simpson's  "A  Treatise  omPlague," 
1905,  by  kind  permission  of  the  Cambridge  University  Press.) 

Hongkong  in  1894  the  death-rate  was  93.4  per  cent,  for  Chinese,  77 
per  cent,  for  Indians,  60  per  cent,  for  Japanese,  100  per  cent,  for 
Eurasians,  and  18.2  per  cent,  for  Europeans.  It  affects  both  men 
and  animals,  and  is  characterized  by  sudden  onset,  high  fever,  pros- 
tration, delirium,  and  the  occurrence  of  exceedingly  painful  lym- 
phatic swellings — buboes — affecting  chiefly  the  inguinal  nodes, 
though  not  infrequently  the  axillary,  and  sometimes  the  cervical, 
nodes.  Death  comes  on  in  severe  cases  in  forty-eight  hours.  The 
pneumonic  form  is  most  rapidly  fatal.  The  longer  the  duration  of 
the  disease,  the  better  the  prognosis.  Autopsy  in  fatal  cases  re- 
veals the  characteristic  enlargement  of  the  lymphatic  nodes,  whose 
contents  are  soft  and  sometimes  purulent. 

Wyman,*    in    his    very    instructive    pamphlet,    "The    Bubonic 
*  Government  Printing  Office,  Washington,  D.  C.,  1900. 


Specific  Organism  545 

Plague,"  finds  it  convenient  to  divide  plague  into  (a)  bubonic  or 
ganglionic,  (b)  septicemic,  and  (c)  pneumonic  forms.  Of  these, 
the  bubonic  form  is  most  frequent  and  the  pneumonic  form  most 
fatal. 

Specific  Organism. — The  bacillus  of  bubonic  plague  was  inde- 
pendently discovered  by  Yersin*  and  Kitasatof  in  the  summer  of 
1894,  during  an  epidemic  of  the  plague  then  raging  at  Hongkong. 
There  seems  to  be  little  doubt  but  that  the  micro-organisms  de- 
scribed by  the  two  observers  are  identical. 

OgataJ  states  that  while  Kitasato  found  the  bacillus  in  the 
blood  of  cadavers,  Yersin  seldom  found  it  in  the  blood,  but  always 
in  the  enlarged  lymphatic  glands;  that  Kitasato's  bacillus  retains 
the  color  when  stained  by  Gram's  method;  Yersin's  does  not;  that 
Kitasato's  bacillus  is  motile;  Yersin's  non-motile;  that  the  colonies 
of  Kitasato's  bacillus,  when  grown  upon  agar,  are  round,  irregular, 


Fig.  225. — Bacillus  of  bubonic  plague  (Yersin). 

grayish  white,  with  a  bluish  tint,  and  resemble  glass-wool  when 
slightly  magnified;  those  of  Yersin's  bacillus,  white  and  transparent, 
with  iridescent  edges.  Ogata,  in  his  investigations,  found  that 
the  bacillus  corresponded  with  the  description  of  Yersin  rather  than 
that  of  Kitasato,  and  it  is  certain  that  of  the  two  the  description 
given  by  Yersin  is  the  more  correct. 

In  the  "Japan  Times,"  Tokio,  November  28,  1899,  Kitasato 
explains  that,  his  investigations  being  made  upon  cadavers  that 
were  partly  putrefied,  he  was  led  to  believe  that  the  bacillus  first 
invaded  the  blood.  Later  studies  upon  living  subjects  showed  him 
the  error  of  this  view  and  the  correctness  of  Yersin's  observation 
that  the  bacilli  first  multiply  in  the  lymphatics. 

Both  Kitasato  and  Yersin  showed  that  in  blood  drawn  from  the 

*  "Ann.  de  PInst.  Pasteur,"  1894,  9. 

t  Preliminary  notice  to  the  bacillus  of  bubonic  plague,  Hongkong,  July  7,  1894. 
j  "  Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Sept.  6,  1897,  Bd.  xxii,  Nos.  6  and  7,  P. 
170. 

35 


546  Plague 

§ . 
finger-tips  and  in  the  softened  contents  of  the  buboes  the  bacillus 

may  be  demonstrable. 

Morphology. — The  bacillus  is  quite  variable.  Uusally  it  is 
short  and  thick — a  "  coco-bacillus,"  as  some  call  it — with  rounded 
ends.  Its  size  is  small  (1.5  to  2  ju  in  length)  and  0.5  to  0.75  /x  in 
breadth.  It  not  infrequently  occurs  in  chains  of  four  or  six  or  even 
more,  and  is  occasionally  encapsulated.  It  shows  active  Brownian 
movements,  which  probably  led  Kitasato  to  consider  it  motile. 
Yersin  did  not  regard  it  as  motile,  and  was  correct.  Gordon* 
claims  that  some  of  the  bacilli  have  flagella.  No  spores  are  formed. 

Staining. — It  stains  by  the  usual  methods;  not  by  Gram's  method. 
When  stained,  the  organism  rarely  appears  uniformly  colored,  be- 
ing darker  at  the  ends  than  at  the  center,  so  as  to  resemble  a  dumb- 
bell or  diplococcus.  The  bacilli  sometimes  appear  vacuolated, 


Fig.  226. — Bacilli  of  plague  and  phagocytes,   from  human  lymphatic    gland 

X  800  (Aoyama).  v 

and  nearly  all  cultures  show  a  variety  of  involution  forms.  Kitasato 
has  compared  the  general  appearance  of  the  bacillus  to  that  of 
chicken-cholera. 

Involution  forms  on  partly  desiccated  agar-agar  not  containing 
glycerin  are  said  by  Haffkine  to  be  characteristic.  The  microbes 
swell  and  form  large,  round,  oval,  pea-shaped,  spindle-shaped  or 
biscuit-like  bodies  which  may  attain  twenty  times  the  normal 
size,  and  gradually  lose  the  ability  to  take  the  stain.  Such  involu- 
tion forms  are  not  seen  in  liquid  culture. 

Cultivation. — Pure  cultures  may  be  from  the  blood  or  from  the 
softened  contents  of  the  buboes,  and  develop  well  upon  artificial 
media.     The  optimum  temperature  is  about  3O°C.     The  extremes 
at  which  growth  occurs  are  20°  and  38°C. 
*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  June  24,  1897,  Bd.  xxi,  Nos.  20  and  21. 


Bouillon  547 

Bouillon. — In  bouillon  a  diffuse  cloudiness  was  observed  by 
Kitasato,  though  Yersin  observed  that  the  cultures  resembled  ery- 
sipelas cocci,  and  contained  zooglea  attached  to  the  sides  and  at 
the  bottom  of  the  tube  of  nearly  clear  fluid. 


Fig.  227. — Bacillus  pestis.  Highly  virulent  culture  forty-eight  hours 
old,  from  the  spleen  of  a  rat.  Unstained  preparation  (Kolle  and 
Wassermann) . 

Haffkine*  found  that  when  an  inoculated  bouillon  culture  is 
allowed  to  stand  perfectly  at  rest,  on  a  firm  shelf  or  table,  a  char- 
acteristic appearance  develops.  In  from  twenty-four  to  forty- 
eight  hours,  the  liquid  remaining  limpid,  flakes  appear  underneath 


^ 


Fig.  228. — Bacillus   pestis.     Involution   forms   from   a   pure  culture  on  3  per 
cent,  sodium  chlorid  agar-agar.     Methylene-blue  (Kolle  and  Wassermann). 

the  surface,  forming  little  islands  of  growth,  which  in  the  next 
twenty-four  to  forty-eight  hours  grow  into  a  jungle  of  long  stalactite- 
like  masses,  the  liquid  remaining  clear.  In  from  four  to  six  days 
these  islands  become  still  more  compact.  If  the  vessels  be  dis- 

*  "Brit.  Med.  Jour.,"  June  12,  1897,  p.  1461. 


548  Plague 

turbed,  they  fall  like  snow  and  are  deposited  at  the  bottom,  leaving 
the  liquid  clear. 

Colonies. — Upon  gelatin  plates  at  22°C.  the  colonies  may  be 
observed  in  twenty-four  hours  by  the  naked  eye.  They  are  pure 
white  or  yellowish  white,  spheric  when  deep  in  the  gelatin,  flat  when 
upon  the  surface,  and  are  about  the  size  of  a  pin's  head.  The 
gelatin  is  not  liquefied.  Upon  microscopic  examination  the  borders 
of  the  colonies  are  found  to  be  sharply  defined.  The  contents  be- 
come more  granular  as  the  age  increases.  The  superficial  colonies 
are  occasionally  surrounded  by  a  fine,  semi-transparent  zone. 

Klein*  says  that  the  colonies  develop  quite  readily  upon  gelatin 
made  from  beef  bouillon  (not  infusion),  appearing  in  twenty-four 
hours,  at  2o°C.,  as  small,  gray,  irregularly  rounded  dots.  Magnifica- 
tion shows  the  colonies  to  be  serrated  at  the  edges  and  made  up  of 


Fig.  229. — Stalactite  growth  of  bacillus  pestis  in  bouillon.  (Reproduced 
from  Simpson's  "A  Treatise  on  Plague,"  1905,  by  kind  permission  of  the  Cam- 
bridge University  Press.) 

i 

short,  oval,  sometimes  double  bacilli.  Some  colonies  contrast 
markedly  with  their  neighbors  in  that  they  are  large,  round,  or  oval, 
and  consist  of  longer  or  shorter,  straight  or  looped  threads  of  bacilli. 
The  appearance  was  much  like  that  of  the  young  colonies  of  Proteus 
vulgaris.  At  first  these  were  regarded  as  contaminations,  but  later 
their  occurrence  was  regarded  as  characteristic  of  the  plague  bacillus. 
The  peculiarities  of  these  colonies  cannot  be  recognized  after  forty- 
eight  hours. 

Gelatin  Punctures. — In  gelatin  puncture  cultures  the  develop- 
ment is  scant.  The  medium  is  not  liquefied;  the  growth  takes  place 
in  the  form  of  a  fine  duct,  little  points  being  seen  on  the  surface 
and  in  the  line  of  puncture.  Sometimes  fine  filaments  project  into 
the  gelatin  from  the  central  puncture. 

Abel  found  the  best  culture-medium  to  be  2  per  cent,  alkaline 

*"Centralbl.  f.  Bakt.  u.  Parasitenk.,"  July  10,  1897,  xxi,  Nos.  24  and  25. 


Vital  Resistance  549 

peptone  solution  containing  i  or  2  per  cent,  of  gelatin,  as  recom- 
mended by  Yersin  and  Wilson. 

Agar-agar. — Upon  agar-agar  the  bacilli  grow  freely,  but  slowly, 
the  colonies  being  whitish  in  color,  with  a  bluish  tint  by  reflected 
light,  and  first  appearing  to  the  naked  eye  when  cultivated  from  the 
blood  of  an  infected  animal  after  about  thirty-six  hours'  incubation 
at  37°C.  Under  the  microscope  they  appear  moist,  with  rounded 
uneven  edges.  The  small  colonies  are  said  to  resemble  tufts  of 
glass-wool.  Microscopic  examination  of  the  agar-agar  culture 
shows  the  presence  of  chains  resembling  streptococci. 

Upon  glycerin-agar  the  development  of  the  colonies  is  slower, 
though  in  the  end  the  colonies  attain  a  larger  size  than  those  grown 
upon  plain  agar. 

Hankin  and  Leumann*  recommended,  for  the  differential  diagnosis 
of  the  plague  bacillus,  a  culture-medium  prepared  by  the  addition 
of  2.5  to  3.5  per  cent,  of  salt  to  ordinary  culture  agar-agar.  When 
transplanted  from  ordinary  agar-agar  to  the  salt  agar-agar,  the  in- 
volution forms  so  characteristic  of  the  bacillus  occur  with  ex- 
ceptional rapidity.  In  bouillon  containing  this  high  percentage  of 
salt  the  stalactite  formation  is  beautiful  and  characteristic. 

Blood-serum. — Upon  blood-serum,  growth,  at  the  temperature 
of  the  incubator,  is  luxuriant  and  forms  a  moist  layer,  of  yellowish- 
gray  color,  unaccompanied  by  liquefaction  of  the  serum. 

Potato. — Upon  potato  no  growth  occurs  at  ordinary  temperatures. 
When  the  potato  is  stood  in  the  incubator  for  a  few  days  a  scanty, 
dry,  whitish  layer  develops. 

Vital  Resistance. — Kitasato  found  that  the  plague  bacillus  did 
not  seem  able  to  withstand  desiccation  longer  than  four  days;  but 
Rappaportf  found  that  they  remained  alive  when  kept  dry  upon 
woolen  threads  at  2o°C.  for  twenty-three  days,  and  Yersin  found 
that  although  it  could  be  secured  from  the  soil  beneath  an  infected 
house  at  a  depth  of  4  to  5  cm.,  the  virulence  of  such  bacilli  was 
lost. 

Kitasato  found  that  the  bacillus  was  killed  by  two  hours'  ex- 
posure to  0.5  per  cent,  carbolic  acid,  and  also  by  exposure  to  a 
temperature  of  8o°C.  for  five  minutes.  Ogata  found  the  bacillus 
instantly  killed  by  5  per  cent,  carbolic  acid,  and  in  fifteen  minutes 
by  0.5  per  cent,  carbolic  acid.  In  o.i  per  cent,  sublimate  solution 
it  is  killed  in  five  minutes. 

According  to  Wyman,  the  bacillus  is  killed  by  exposure  to  55°C. 
for  ten  minutes.  The  German  Plague  Commission  found  that  the 
bacilli  were  killed  by  exposure  to  direct  sunlight  for  three  or  four 
hours;  and  BowhillJ  found  that  they  are  killed  by  drying  at  ordinary 
room  temperatures  in  about  four  days. 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Oct.,  1897,  Bd.  xxii,  Nos.  16  and  17,  p. 
438. 

t  Quoted  by  Wyman. 

j  "  Manual  of  Bacteriological  Technique  and  Special  Bacteriology,"  1899,  p.  197. 


550  Plague 

Wilson*  found  the  thermal  death-point  of  the  organism  one  or 
two  degrees  higher  than  that  of  the  majority  of  non-sporulating 
pathogenic  bacteria,  and  that  the  influence  of  sunlight  and  desicca- 
tion cannot  be  relied  upon  to  destroy  it. 

Rosenauj  found  temperature  the  most  important  factor,  as  it 
dies  quickly  when  kept  dry  at  37°C.,  but  remains  alive  for  months 
when  kept  dry  at  i9°C.  Sunlight  kills  it  in  a  few  hours.  A  tem- 
perature of  7o°C.  is  invariably  fatal  in  a  short  time. 

Metabolism. — The  bacillus  develops  best  under  aerobic  con- 
ditions though  it  develops  to  a  slight  extent  also  under  anaerobic 
conditions.  In  glucose-containing  media  it  does  not  form  gas. 
No  indol  is  formed.  Ordinarily  the  culture-medium  is  acidified, 
the  acid  reaction  persisting  for  three  weeks  or  more. 

Ghon,J  Wernicke,§  and  others  who  have  studied  the  toxic 
products  of  the  bacillus  all  incline  to  the  belief  that  it  forms  only 
endotoxin. 

Kossee  and  Overbeck,||  however,  believe  that  there  is,  in  addition, 
a  soluble  exotoxin  that  is  of  importance. 

Bielonovsky**  finds  that  broth,  agar,  and  serum  cultures  of  the 
plague  bacillus  possess  the  property  of  hemolyzing  the  blood  .of 
normal  animals.  The  hemolytic  power  of  filtrates  of  plague  cultures 
increases  up  to  the  thirteenth  or  fourteenth  day,  then  gradually  di- 
minishes, but  without  completely  disappearing.  The  hemolysins 
are  notably  resistant  to  heat,  not  being  destroyed  below  ioo°C. 

Experimental  Infection. — Mice,  rats,  guinea-pigs,  rabbits,  and 
monkeys  are  all  susceptible  to  experimental  inoculation.  When 
blood,  lymphatic  pulp,  or  pure  cultures  are  inoculated  into  them, 
the  animals  become  ill  in  from  one  to  two  days,  according  to  their 
size  and  the  virulence  of  the  bacillus.  Their  eyes  become  watery, 
they  show  disinclination  to  take  food  or  to  make  any  bodily  effort, 
the  temperature  rises  to  4i.5°C.,  they  remain  quiet  in  a  corner  of 
the  cage,  and  die  with  convulsive  symptoms  in  from  two  to  seven 
days.  If  the  inoculation  be  made  intravenously,  no  lymphatic 
enlargement  occurs;  but  if  it  be  made  subcutaneously,  the  nearest 
lymph  nodes  always  enlarge  and  suppurate  if  the  animal  live  long 
enough.  The  bacilli  are  found  everywhere  in  the  blood,  but  not  in 
very  large  numbers. 

Rats  suffer  from  both  an  acute  septicemic  and  a  chronic  form  of 
the  disease.  In  the  former  an  infiltration  or  watery  edema  can  be 
observed  in  a  few  hours  about  the  point  of  inoculation.  The  autopsy 
shows  the  infiltration  to  be  made  up  of  a  yellowish  gelatinous  exuda- 

*  "Journal  of  Medical  Research,"  July,  1901,  vol.  vi,  No.  i,  p.  53. 
t  Bulletin  No.  4  of  the  Hygienic  Laboratory  of  the  U.  S.  Marine  Hospital 
Service,  1901. 
t  Wien,  1898. 

§  "Gentralbl.  f.  Bakt.,"  etc.,  1898,  xxrv. 
||  "  Arbeiten  aus  d.  Kaiserl.  Gesundheitsamte,"  1901,  xvin. 
**  "Arch,  des  Sci.  Biol.,"  Petersb.,  1904.     St.  Tome  x,  No.  4. 


Mode  of  Infection  551 

tion.  The  spleen  and  liver  are  enlarged,  the  former  often  pre- 
senting an  appearance  similar  to  that  observed  in  miliary  tuber- 
culosis. Sometimes  there  is  universal  enlargement  of  the  lymphatic 
glands.  Bacilli  are  found  in  the  blood  and  in  all  the  internal  organs. 
Skin  eruptions  may  occur  during  life,  and  upon  the  inner  abdominal 
walls  petechise  and  occasional  hemorrhages  may  be  found.  The 
intestine  is  hyperemic,  the  adrenals  congested.  Serosanguinolent 
effusions  may  occur  into  the  serous  cavities. 

In  the  latter,  they  sometimes  have  encapsulated  caseous  nodules  in 
the  submaxillary  glands,  caseous  bronchial  glands,  and  fibroid 
pneumonia,  months  after  infection.  In  all  such  cases  virulent 
plague  bacilli  are  present. 

In  and  about  San  Francisco  the  extermination  of  rats  for  the 
eradication  of  the  plague  was  unexpectedly  complicated  by  the 
discovery  that  other  rodents  with  which  the  rats  came  into  contact 
also  harbored  the  plague  bacilli.  McCoy  and  Smith*  found  this 
to  be  true  of  the  prairie  dog,  the  desert  wood  rat,  the  rock  squirrel, 
and  the  brush  rat.  To  insure  security  against  the  recurrence  of 
the  disease  among  men  necessitates  continued  observation  of  these 
animals  and  the  extermination  of  diseased  colonies,  as  well  as  their 
complete  extermination  in  the  neighborhood  of  human  habitations. 

Devellj  has  found  frogs  susceptible  to  the  disease. 

Mode  of  Infection. — The  plague  bacillus  may  enter  the  body  by 
inhalation,  from  an  atmosphere  through  which  it  is  disseminated, 
under  which  circumstances  it  usually  causes  the  pneumonic  type 
of  the  disease  which  is  not  unlike  other  forms  of  pneumonia.  The 
lung  is  consolidated,  enormous  numbers  of  plague  bacilli  occur  in 
the  sputum,  the  fever  is  high,  and  death  occurs  in  a  few  days. 

Plague  pneumonia  does  not  necessarily  imply  infection  through 
inhalation  of  the  bacilli,  however,  for  it  occasionally  occurs  as  a 
complication  in  the  bubonic  form  of  the  disease. 

Klein  found  that  animals  fed  upon  cultures  of  the  bacillus  or 
upon  the  flesh  of  animals  dead  of  the  disease  became  ill  and  died 
with  typical  symptoms.  Simond  has  confirmed  his  results  and 
it  is  not  improbable  that  the  disease  is  sometimes  acquired  by 
rats  through  feeding  upon  their  companions  that  have  died  of  it. 
The  micro-organisms  seem  able  to  penetrate  any  of  the  mucous  mem- 
branes, so  that  infection  usually  follows  their  application  to  the  un- 
injured conjunctiva,  nasal,  buccal,  vaginal  or  gastro-intestinal 
surfaces. 

Cutaneous  and  Subcutaneous  Inoculation. — All  susceptible  ani- 
mals quickly  become  infected  if  a  needle  infected  with  'a  culture 
of  the  bacilli  or  with  material  from  a  bubo  or  other  infective  lesion 
be  used  to  puncture  or  scratch  the  skin.  Wyssokowitsch  and 
Zabolotnyt  found  monkeys  highly  susceptible  to  plague,  especially 

*  "Journal  of  Infectious  Diseases,"  1910,  vii,  p.  374. 
t  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Oct.  12,  1897. 


552 


Plague 


when  subcutaneously  inoculated.  When  an  inoculation  was  made 
with  a  pin  dipped  in  a  culture  of  the  bacillus,  the  puncture  being 
made  in  the  palm  of  the  hand  or  sole  of  the  foot,  the  monkeys  always 
died  in  from  three  to  seven  days.  In  these  cases  the  local  edema 
observed  by  Yersin  did  not  occur.  They  point  out  the  interest 
attaching  to  infection  through  so  insignificant  a  wound  and  without 
local  lesions.  Weichselbaum,  Albrecht  and  Gohn  have  found  that 
rats  may  be  infected  by  rubbing  the  infective  material  upon  the 
surface  of  the  shaved  skin,  the  method  being  employed  for  making 
a  diagnosis  of  the  disease  in  suspected  cases.  Rats  and  mice  in- 


Antepygidial  bristle 
Pygidium 


Stigmata  . 
Penis  ... 


Abdomen 


Thorax       Head 


Antenna 
Eye 

Ocular  bristle 


Oral  bristle 
-    Maxillary  palp 
Maxilla 


"••  Hip  or  coxa 

•••    Trochanter 

—    Femur 


Fig.  230.— Xenopsylla  cheopis  (male)  (from  Rothschild).  v 

fected  through  the  skin  usually  die  in  two  or  three  days,  guinea- 
pigs  in  two  to  five  days,  rabbits  in  three  to  eight  days. 

The  facility  with  which  dermal  infection  could  be  brought  about, 
quickly  suggested  that  the  skin  might  be  the  common  route,  and 
that  biting  insects  might  act  as  vectors. 

Yersin  showed  that  flies  taking  up  the  bacilli  may  die  of  the  in- 
fection. Macerating  and  crushing  a  fly  in  bouillon,  he  not  only 
succeeded  in  obtaining  the  bacillus,  but  infected  an  animal  with  it. 

Nuttall,*  in  repeating  Yersin's  fly  experiment,  found  his  observa- 
tion correct,  and  showed  that  flies  fed  with  the  cadavers  of  plague- 
infected  mice  die  in  a  variable  length  of  time.  Large  numbers  of 
plague  bacilli  were  found  in  their  intestines.  He  also  found  that 
bed-bugs  allowed  to  prey  upon  infected  animals  took  up  large 
*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  xxi,  No.  24,  Aug.  13,  1897. 


Mode  of  Infection  553 

numbers  of  the  plague  bacilli  and  retained  them  for  a  number  of 
days.  These  bugs  did  not,  however,  infect  healthy  animals  when 
allowed  to  bite  them;  but  Nuttall  was  not  satisfied  that  the  number 
of  his  experiments  upon  this  point  was  great  enough  to  prove  that 
plague  cannot  be  thus  spread.  Vergbitski,*  however,  was  more 
successful  and  a  bed-bug  that  he  caused  to  bite  a  patient  suffering 
from  plague,  subsequently  transmitted  the  disease  to  a  rat.  It  is 
quite  possible  that  mosquitoes  and  biting  flies  may  transmit  it. 
As  epidemics  of  human  plague  are  commonly  preceded  by  epi- 
demics among  the  rats  which  die  in  great  numbers,  it  early  became 
a  question  whether  the  plague  among  them  was  not  caused  by  the 
bites  of  fleas,  and  whether  it  might  not  also  be  fleas  that  infected 
man. 

M.  Herzogf  has  shown  that  pediculi  may  harbor  plague  bacilli 
and  act  as  carriers  of  the  disease. 

Ogata  found  plague  bacilli  in  fleas  taken  from  diseased  rats. 
He  crushed  some  fleas  between  sterile  object-glasses  and  introduced 
the  juice  into  the  subcutaneous  tissues  of  a  mouse,  which  died 
in  three  days  with  typical  plague,  a  control-animal  remaining  well. 
Some  guinea-pigs  taken  for  experimental  purposes  into  a  plague 
district  died  spontaneously  of  the  disease,  presumably  because  of 
flea  infection. 

Galli-Valerio{  and  others  think  that  the  fleas  of  the  mouse  and 
rat  are  incapable  of  living  upon  man  and  do  not  bite  him,  and 
that  it  is  only  the  Pulex  irritans,  or  human  flea,  that  can  transmit 
the  disease  from  man  to  man.  Tidswell,§  however,  found  that 
of  100  fleas  collected  from  rats — there  were  four  species,  of  which 
three — the  most  common  kinds — bit  men  as  well  as  rats.  Lisbon|| 
found  that  of  246  fleas  caught  on  men  in  the  absence  of  plague,  only 
one  was  a  rat  flea,  but  out  of  30  fleas  caught  upon  men  in  a  lodging- 
house,  during  plague,  14  were  rat  fleas.  This  seems  to  show  that 
as  the  rats  die  off  their  fleas  seek  new  hosts,  and  may  thus  contribute 
to  the  spread  of  the  disease. 

That  fleas  can  cause  the  transmission  of  plague  from  animal 
to  animal  has  been  proved  by  experiments  made  in  India.  These 
experiments,  which  are  published  as  "  Reports  on  Plague  Investiga- 
tions in  India,"  issued  by  the  Advisory  Committee  appointed  by 
the  Secretary  of  State  for  India,  the  Royal  Society,  and  the  Lister 
Institute,  appear  in  the  "Journal  of  Hygiene"  from  1906  onward.** 
It  seems  from  these  experiments  that  human  fleas  (Pulex  irritans) 
do  not  bite  rats,  but  that  the  rat  fleas  of  all  kinds  do,  though  not 

*  "Jour-  of  Hygiene,"  1904,  vra,  185. 
"Amer.  Jour.  Med.  Sci.,"  March,  1895. 
Ibid.,  xxvii,  No.  i,  p.  i,  Jan.  6,  1900. 
"British  Medical  Journal,"  June  27,  1903. 
"Times  of  India,"  Nov.  26,  1904. 
**  "Journal  of  Hygiene,"  Sept.,  1906,  vol.  vi,  p.  421;  July,  1907,  vol.  vn,  p.  324; 
Dec.,  1907,  vol.  vn,  p,  693;  May,  1908,  vol.  vm,  p.  162;  1909,  vol.  ix;  1910,  vol. 
x;  1911,  vol.  xi. 


554  Plague 

willingly,  bite  men.  By  placing  guinea-pigs  in  cages  upon  the  floor 
of  the  infected  houses,  the  fleas  of  all  kinds  quickly  attack  them 
with  resulting  infection,  but  if  the  guinea-pigs  are  kept  in  flea- 
proof  cages,  or  if  the  cages  are  surrounded  by  "  tangle-foot,"  or 
"sticky  fly-paper,"  the  fleas,  not  being  able  to  spring  over  the 
barrier,  are  caught  on  the  sticky  surf  aces  and  do  not  reach  the  guinea- 
pigs,  which  then  remain  uninfected.  What  is  true  of  the  guinea- 
pigs  is  undoubtedly  true  of  the  rats;  the  disease  is  transmitted  from 
rat  to  rat  by  the  fleas.  When  the  rats  die,  the  fleas  being  hungry, 
jump  upon  any  convenient  warm-blooded  animal  to  satisfy  their 
appetites,  and  when  human  beings  become  their  victims,  infection 
may  follow  the  bites.  It  is  now  clearly  demonstrated  that  though 
Pulex  irritans,  the  human  flea,  prefers  to  bite  human  beings,  and 
Xenopsylla  cheopis,  the  rat  flea,  prefers  to  bite  rats,  under  stress  of 
necessity  preferences  are  set  aside  and  miscellaneous  feeding  prac- 
tised by  these  and  probably  all  other  fleas. 

A  peculiar  circumstance  attending  flea  infection  has  been  discovered 
by  Bacot  and  Martin*  who  find  that  when  Xenopsylla  cheopis  and 
Ceratophyllus  fascia  tus  are  fed  upon  septicemic  plague  blood,  the  re- 
spective fleas  suffer  from  a  temporary  obstruction  at  the  entrance 
of  the  stomach,  caused  by  a  massive  growth  of  the  plague  bacilli. 
This  culture  appears  to  start  in  the  intercellular  recesses  of  the 
proventriculus  and  grows  so  abundantly  as  to  choke  this  organ  and 
extend  into  the  esophagus.  Fleas  in  this  condition  are  not 
prevented  from  sucking  blood,  as  the  pump  is  in  the  pharynx,  but 
they  only  succeed  in  distending  an  already  contaminated  esophagus, 
and  on  the  cessation  of  the  pumping  act,  some  of  the  blood  is 
forced  back  into  the  wound.  Such  fleas  are  persistent  in  their 
endeavors  to  feed  and  this  renders  them  particularly  dangerous. 

Bacotf  found  that  infected  fleas  remained  infectious  when 
starved  for  forty-seven  days,  and  that  when  they  were  subse- 
quently permitted  to  feed  upon  mice,  another  period  of  twenty 
days  might  supervene  before  the  mice  became  infected. 

The  cutaneous  and  subcutaneous  inoculation  in  man  is  followed 
by  lymphatic  invasion  with  bubo  formation.  Beyond  this  lymphatic 
barrier  but  few  bacilli  get  so  that  in  the  greater  number  of  cases 
with  buboes  there  is  little  blood  infection.  However,  should  the 
bacilli  be  highly  virulent  or  the  patient  exceptionally  susceptible, 
the  septicemic  form  of  the  disease  may  supervene,  and  the  case 
progress  to  a  rapidly  fatal  termination. 

Intravenous  and  Intraperitoneal  Inoculations  produce  rapidly  fatal 
septicemic  forms  of  plague. 

KleinJ  found  that  intraperitoneal  injection  of  the  bacillus  into 
guinea-pigs  was  of  diagnostic  value,  producing  a  thick,  cloudy, 

"The  Journal  of  Hygiene,"  Plague  Supplement,  in,  1914,  p.  423. 
t"  Journal  of  Hygiene,"  Plague  Supplement,  No.  rv,  Jan.,  1915,  p.  770. 
j  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  xxi,  No.  24,  July  10,  1897,  p.  849. 


Diagnosis  555 

peritoneal  exudate  rich  in  leukocytes  and  containing  characteristic 
chains  of  the  plague  bacillus,  occurring  in  from  twenty-four  to 
forty-eight  hours. 

Diagnosis. — It  seems  possible  to  make  a  diagnosis  of  the  disease 
in  doubtful  cases  by  examining  the  blood,  but  it  is  admitted  that  a 
good  deal  of  bacteriologic  practice  is  necessary  for  the  purpose. 

Abel  found  that  blood-examinations  may  yield  doubtful  results 
because  of  the  variable  appearance  of  the  contained  bacilli,  which 
may  easily  be  mistaken  for  other  bacteria.  He  deems  the  best 
tests  to  be  the  inoculation  of  broth  cultures  and  the  subsequent 
inoculation  into  animals,  which,  he  advises,  should  have  been  pre- 
viously vaccinated  against  the  streptococcus. 

Kolle*  has  suggested  a  method  valuable  both  for  the  diagnosis 
of  the  disease  and  for  estimating  the  virulence  of  the  bacillus.  It 
is  as  follows:  "The  skin  over  a  portion  of  the  abdominal  wall  of 
the  guinea-pig  is  shaved,  care  being  taken  to  avoid  the  slightest 
injury  of  the  skin.  The  infective  material  is  carefully  rubbed  into 
the  shaved  skin.  Important,  in  order  rightly  to  understand  the 
occurrence  of  plague  infection,  is  the  fact  disclosed  here  in  the  case 
of  guinea-pigs,  that  by  this  method  of  inoculation  the  animals 
present  the  picture  of  true  bubonic  plague — that  is  to  say,  the  pro- 
duction of  nodules  in  the  various  organs,  principally  in  the  spleen. 
In  this  manner  guinea-pigs,  which  would  not  be  affected  by  large 
subcutaneous  injections,  even  amounting  to  2  mg.  of  agar  culture 
(equal  to  a  loop)  of  low-virulence  plague  bacillus,  may  be  infected 
and  eventually  succumb." 

The  postmortem  appearance  of  the.  body  of  a  plague-infected 
rat  is  as  follows:!  Subcutaneous  hemorrhages  occur  in  about  40 
per  cent,  of  the  animals  and  are  most  frequently  to  be  seen  in  the 
submaxillary  region.  Buboes  are  present  in  the  majority  of  cases, 
usually  in  some  one  locality,  and  commonly  about  the  neck.  The 
liver  may  show  necrotic  changes  which  have  the  appearance  of  an 
excessive  deposit  of  fat,  and  a  condition  of  the  greatest  importance 
in  diagnosis  is  the  occurrence  of  small  necrotic  foci  scattered  over 
its  surface  and  throughout  its  substance.  The  spleen  is  firm  and 
does  not  collapse  like  a  soft  normal  spleen;  granules  or  nodules  may 
be  well  marked  in  it  and  may  be  confluent.  The  kidneys  and 
suprarenal  capsules  are  often  congested.  Hemorrhages  are  fairly 
common  in  the  lungs  and  visceral  pleura.  The  presence  of  pleural 
effusion  is  very  characteristic  and  of  great  value  in  diagnosis.  In 
naturally  infected  plague  rats,  the  most  important  features  for 
purposes  of  diagnosis  are: 

1.  A  typical  bubo — most  commonly  in  the  neck. 

2.  Granular  liver — not  seen  except  in  plague  rats. 

*  "See  Havelburg,  "Public  Health  Reports,"  Aug.  15,  1902,  vol.  xvu,  No.  33, 
p.  1863. 

t  See  "Journal  of  Hygiene,"  1907,  vu,  324. 


Plague 

3.  Hemorrhages  beneath  the  skin  and  in  the  internal  organs  are 
very  suggestive. 

4.  Pleural  effusion. 

In  putrid  rats,  bubo,  granular  liver  and  pleural  effusion  may  persist 
and  are  of  great  significance.  A  microscopical  examination  of 
scrapings  from  buboes  and  spleen  and  inoculation  tests  will  clinch 
the  diagnosis  (Besson). 

Virulence. — By  frequent  passage  through  animals  of  the  same 
species  the  bacillus  can  be  much  increased  in  virulence.  Kolle 
recommended  rats  for  this  purpose,  and,  indeed,  declared  that 
without  the  use  of  rats  it  is  impossible  to  keep  cultures  at  a  high 
grade  of  virulence.  According  to  the  researches  of  the  Advisory 
Committee  for  the  study  of  plague  in  India,  this  is  an  error.  The 
virulence  of  plague  bacilli  for  rats  is  subject  to  very  little  change. 
Their  members  in  investigating  the  question  made  twenty-six 
passages  from  rat  to  rat,  by  subcutaneous  inoculation,  during 
eighty-nine  days,  and  found  the  original  virulence  of  the  organism 
unchanged. 

Yersin  found  that  when  cultivated  for  any  length  of  time  upon 
culture-media,  especially  agar-agar,  the  virulence  was  rapidly  lost 
and  the  bacillus  eventually  died.  On  the  other  hand,  when  con- 
stantly inoculated  from  animal  to  animal,  the  virulence  of  the 
bacillus  is  much  increased. 

Knorr,  Yersin,  Calmette,  and  Borrel*  have  shown  that  the 
bacillus  made  virulent  by  frequent  passage  through  mice  is  not  in- 
creased in  virulence  for  rabbits. 

This  no  doubt  depends  upon  the  sensitivity  of  the  bacillus  to 
the  protective  substances  of  the  body  juices,  immunization  against 
those  of  one  animal  not  necessarily  protecting  the  organism  against 
those  of  other  animals. 

Sanitation. — A  disease  that  may  be  transmitted  from  man  to 
man  by  atmospheric  infection  and  inhalation,  that  can  be  transported 
from  place  to  place  by  fomites,  that  occurs  in  epidemic  form  among 
the  lower  animals  as  well  as  among  men,  and  that  can  be  trans- 
mitted from  man  to  man  and  from  lower  animals  to  man  by  biting 
insects,  must  inevitably  become  a  source  of  anxiety  to  the  sanitarian. 

The  preventive  measures  must  take  account  of  men,  rats,  and 
goods.  If  vessels  are  permitted  to  visit  and  leave  plague-stricken 
ports,  means  must  be  taken  to  see  that  all  passengers  are  healthy 
at  the  time  of  leaving  and  have  remained  so  during  the  voyage,  and 
provision  should  be  made  at  the  port  of  entry  for  the  disinfection 
of  the  cargo  before  the  goods  are  landed.  But  the  rats  must  be 
given  special  consideration,  for,  so  soon  as  the  vessel  reaches  port 
some  of  them  jump  overboard  and  swim  to  the  shore,  carrying  the 
disease  with  them.  When  a  vessel  visits  a  plague  port,  every  pre- 
caution should  be  taken  to  prevent  the  entrance  of  rats,  first  by 
*  "Ann.  de  1'Inst.  Pasteur.,"  July,  1895. 


Immunity  557 

anchoring  in  the  stream  instead  of  tying  to  the  dock;  by  carefully 
scrutinizing  the  packages  taken  from  the  lighters  to  see  that  there 
are  no  rats  hidden  among  them;  by  placing  large  metal  shields  or 
reversed  funnels  about  all  anchor  chains,  hawsers,  and  cables  so 
that  no  rats  can  climb  up  from  the  water  in  which  they  are  swim- 
ming at  night.  Arrangements  should  also  be  made  for  rat  destruc- 
tion on  board  the  ship  by  means  of  sulphurous  oxid  or  other  poi- 
sonous vapors  to  rid  the  ship  of  rats  before  the  next  port  is  reached. 
Passengers  and  crew  should  also  be  kept  in  quarantine  before  ming- 
ling with  society.  It  is  much  more  easy  to  keep  plague  out  of  a 
port  than  to  combat  it  when  it  has  entered,  for  under  the  latter 
condition  are  involved  the  isolation  of  the  patients  in  rat-free  and 
vermin-free  quarters,  the  disinfection  of  the  premises  and  goods 
where  the  case  arose,  and  an  immediate  warfare  upon  the  rats  and 
other  small  animals  of  the  neighborhood.  To  emphasize  how 
difficult  the  latter  may  be  it  is  only  necessary  to  point  out  that 
plague  reached  San  Francisco  in  May,  1907,  during  which  year 
there  were  156  cases  and  76  deaths.  Every  precaution  was  taken 
to  prevent  its  spread,  and  though  the  extermination  of  rats  was 
practised  at  great  expense  and  with  the  utmost  thoroughness,  the 
disease  spread  to  the  ground  squirrels  and  other  small  rodents,  and 
in  1914  plague-infected  rodents  were  still  to  be  found  in  the  outskirts 
of  the  city. 

Immunity. — An  attack  of  plague  usually  exempts  from  future 
attacks.  Artificial  immunity  may  therefore  be  induced  in  both  man 
and  the  lower  animals  by  a  variety  of  methods. 

I.  Active  Immunity. — Haffkine*  followed  his  plan  of  preventive 
inoculation  as  employed  against  cholera,  and  has  invented  a  method 
of  prophylaxis  based  upon  the  use  of  devitalized  cultures.  Bouillon 
cultures  are  grown  in  flasks  for  six  weeks;  small  floating  drops  of 
butter  being  employed  to  make  the  "islands"  of  plague  bacilli 
float.  Successive  crops  of  the  island-stalacite  growth  are  pre- 
cipitated by  agitating  the  flasks.  In  this  manner  an  "intense  extra- 
cellular toxin,"  containing  large  numbers  of  the  bacilli  is  prepared. 
After  testing  the  purity  of  the  culture  by  transplantation  to  agar- 
agar,  it  was  killed  by  exposure  to  65°C.  for  one  hour  and  received 
an  addition  of  0.5  per  cent,  of  phenol.  The  preparation  was  used  in 
doses  of  2  to  3  cc.  as  a  preventive  inoculation.  A  more  thorough 
and  prolonged  immunity  resulted  from  the  administration  of  a 
second  dose  ten  days  after  the  first. 

An  interesting  collection  of  statistics,  showing  in  a  convincing 
manner  the  value  of  the  Haffkine  prophylactic,  is  published  by 
Leumann,  of  Hubli.  The  figures,  together  with  a  great  deal  of 
interesting  information  upon  the  subject,  can  be  found  in  the 
paper  upon  "A  Visit  to  the  Plague  Districts  in  India,"  by  Barker 
and  Flint,  t 

*  "Brit.  Med.  Jour.,"  June  12,  1897;  "India  Medical  Gazette,"  1897. 
t  "New  York  Med.  Jour.,"  Feb.  3,  1900. 


558  Plague 

The  German  Plague  Commission*  believed  that  an  important 
improvement  in  the  vaccine  could  be  brought  about  by  the  use  of 
the  method  now  generally  employed  in  making  bacterio-vaccines 
(q.v.).  They  therefore  caused  the  bacilli  to  grow  in  Roux  bottles 
upon  the  surface  of  agar-agar  for  forty-eight  hours,  washed  off  the 
bacteria  with  bouillon  or  physiological  salt  solution  so  that  i  cc.  of 
the  suspension  contained  about  2.5  mg.  of  bacilli,  and  then  heated  the 
suspension  for  an  hour  or  so  at  65°C.  After  heating,  0.5  per  cent, 
of  phenol  was  added.  This  mode  of  preparation  has  the  advantage 
of  excluding  the  possibility  of  the  accidental  growth  of  tetanus  bacilli 
and  other  micro-organisms  in  the  culture.  The  vaccine  appeared  to 
give  excellent  results  in  Brazil  where  it  was  extensively  used. 
Haffkine,  however,  considers  his  method  preferable  because  of  the 
greater  quantity  of  immunizing  metabolic  products  of  the  bacilli 
contained  in  the  fluid  cultures  on  account  of  their  prolonged  growth. 

The  immunity  conferred  by  the  Haffkine  prophylactic  is  supposed 
to  last  about  a  year.  The  preparation  must  never  be  used  if  the 
person  has  already  been  exposed  to  infection,  and  is  in  the  incuba- 
tion stage  of  the  disease,  as  it  contains  the  toxins  of  the  disease,  and 
therefore  greatly  intensifies  the  existing  condition.  When  injected 
into  healthy  persons  it  always  produces  some  fever,  slight  local 
swellings,  and  malaise. 

Kolle  and  Ottof  from  experimental  studies  of  plague  immunity 
in  rats,  came  to  the  conclusion  that  a  prophylactic  injection  con- 
sisting of  a  culture  of  attenuated  plague  bacilli  would  have  a  much 
more  powerful  and  lasting  effect  than  one  consisting  of  a  killed 
bacilli.  The  same  conclusion  was  reached  by  Kolle  and  Strong! 
and  the  first  use  of  living  cultures  for  preventive  inoculation  in 
human  beings  was  byStrong§who  found  them  to  be  devoid  of  danger, 
and  is  hopeful  regarding  their  efficacy. 

Besredka||  advises  the  use  of  a  killed  culture  sehsitized  by  the 
application  of  immune  serum.  Such  vaccine  seems  to  be  productive 
of  long  enduring  immunity  when  tried  upon  experimental  animals. 

Rowland**  is  under  the  impression  that  the  essential  immunizing 
antigen  is  in  the  bacterial  nucleoproteins.  These  he  extracts  from 
the  bacterial  cells  by  treating  them  while  moist  with  anhydrous 
sodium  sulphate,  freezing,  permitting  the  water  to  be  absorbed  by 
the  chemical,  thawing,  and  then  filtering  off  the  fluid  at  37°C.  The 
filtrate  thus  obtained  is  highly  toxic,  fatal  to  rats  in  minute  doses  and 
capable  of  effecting  immunization. 

II.  Passive  Immunity  against  plague,  through  the  employment 

*  "Arbeiten  aus  dem  Kaiserl.  Gesundheitsamte,"  1899,  xvi. 
t" Deutsche  med.  Wochenschrift,"  1903,  p.  493;  "Zeitschrift  fur  Hygiene," 
1903,  XLV,  507. 

"Deutsche  med.  Wochenschrift,"  1906,  xxxii,  413. 
"Jour.  Medical  Research,"  N.  S.,  1908,  xvm,  325. 
"Bull,  de  1'Inst.  Pasteur,"  1910,  vin,  241. 
!*  "Jour,  of  Hygiene,"  1912,  xu,  344. 


The  Plague  Fleas  559 

of  the  serums  of  experimentally  immunized  animals  for  hypo- 
dermatic injection  into  man  was  tried  soon  after  the  discovery  of 
the  plague  bacillus.  Kitasato's  experiments  first  showed  that  it 
was  possible  to  bring  about  immunity  against  the  disease,  and 
Yersin,  working  in  India,  and  Fitzpatrick,  in  New  York,  have 
successfully  immunized  large  animals  (horses,  sheep,  and  goats). 
The  serum  of  the  immunized  animals  contains  specific  agglutinins 
and  bacteriolysins  as  well  as  an  antitoxin,  capable  not  only  of  pre- 
venting the  disease,  but  also  of  curing  it  in  mice  and  guinea-pigs 
and  probably  in  man. 

Study  of  plague  serums  has  been  conducted  by  Yersin,  Calmette 
and  Borrel,*  but  their  value  as  a  prophylactic  lacks  demonstration. 

Wyssokowitsch  and  Zabolotny,f  used  96  monkeys  in  the 
study  of  the  value  of  the  "plague  serums,"  and  found  that  when 
treatment  was  begun  within  two  days  from  the  time  of  inoculation 
the  animals  could  be  saved,  even  though  symptoms  of  the  disease 
were  marked.  After  the  second  day  the  treatment  could  be  relied 
upon.  The  dose  necessary  was  20  cc.  of  a  serum  having  a  potency 
of  i  :  10.  If  too  little  serum  was  given,  the  course  of  the  disease  was 
retarded  and  the  animal  improved  for  a  time,  then  suffered  a  re- 
lapse, and  died  in  from  thirteen  to  seventeen  days.  The  serum  also 
produced  immunity,  but  of  only  ten  to  fourteen  days'  duration. 
Immunity  lasting  three  weeks  was  conferred  by  inoculating  a  monkey 
with  an  agar-agar  culture  heated  to  6o°C.  If  too  large  a  dose  of 
such  a  culture  was  given,  however,  the  animal  was  enfeebled  and 
remained  susceptible. 

THE  PLAGUE  FLEAS 

Fleas  were  formerly  classed  as  a  suborder  of  the  Diptera,  or  two-winged  in- 
sects, and  because  they  had  no  wings,  were  known  as  Aphaniptera.  At  the 
present  time  they  constitute  an  order  by  themselves,  the  Siphonaptera. 

Every  flea  undergoes  a  complete  metamorphosis.  It  begins  its  life  history  as 
a  minute,  oval,  pearly-white  egg  measuring  about  0.6  mm.  in  length,  that  falls 
from  the  body  of  the  female  to  the  floor  or  ground.  The  eggs  of  fleas  are  not 
cemented  to  the  hairs  like  those  of  lice,  but  drop  to  the  ground  where  the  larva 
lives.  More  or  less  eggs  are  therefore  always  scattered  about  where  dogs,  cats, 
rats,  mice  or  other  animals  that  harbor  fleas  are  to  be  found,  and  more  or  less 
larvae  and  pupae  are  likewise  to  be  found  in  such  places.  In  the  course  of  from 
five  to  ten  days,  a  minute,  active  caterpillar-like  larva  emerges  from  the  egg  to 
feed  upon  such  organic  matter  as  it  may  find  for  the  six  to  eight  weeks  of  this 
stage.  During  the  larval  period  the  skin  is  shed  three  or  four  times.  When  full 
grown,  the  larva  empties  its  alimentary  canal,  spins  itself  a  tiny  silken  cocoon, 
sometimes  including  minute  bits  of  rubbish  or  grains  of  sand  in  its  structure, 
sheds  its  skin  for  the  last  time,  and  becomes  a  pupa.  As  such  it  is  inactive  for 
from  two  to  eight  weeks,  according  to  external  conditions  of  temperature  and 
moisture,  then  opens  the  cocoon  and  emerges  from  the  pupa  shell,  a  perfect  in- 
sect— the  flea  proper. 

The  adult  fleas,  both  males  and  females,  have  soft  exoskeletons  at  first,  but 
soon  they  harden,  through  the  formation  of  chitin,  to  the  well-known  tough  and 
brittle  armor. 

The  male  differs  from  the  female  in  being  smaller  and  in  its  shorter  abdomen. 

*  "Ann.  de  1'Inst.  Pasteur,"  1895,  ix,  589. 
t  Loc.  cit. 


560 


Plague 


Both  insects  hop  about  in  search  of  the  appropriate  warm-blooded  hosts  upon 
whose  blood  they  are  to  live.  Each  kind  of  flea  has  a  preferred  host,  but  the 
tastes  of  all  are  more  or  less  cosmopolitan,  so  that  in  the  absence  of  the  preferred 
host,  another  kind  of  warm-blooded  creature  will  do.  Adult  fleas  live  solely  by 
sucking  blood. 

The  longevity  of  a  flea  varies  according  to  conditions  of  temperature  and  mois- 
ture Life  is  longest  when  the  temperature  is  high  and  the  ground  not  too  dry. 
They  may  live  for  months  without  feeding;  when  regularly  fed  they  can  live  at 
least  a  year  and  a  half.  The  longevity  of  the  fleas  in  the  adult  stage,  the  long 
periods  of  abstention  from  food  that  they  may  suffer  without  dying,  and  the  ac- 
cessions to  their  numbers  that  may  occur  through  the  perfection  of  their  embry- 
onal fellows  in  the  same  place,  explain  why  families  returning  to  their  closed  city 
houses,  or  going  to  their  closed  country  houses,  sometimes  find  them  after  months 
of  desertion,  occupied  by  a  welcoming  host  of  fleas.  They  are  the  progeny  of 


r 


MiTOSrs 
/MJ^n 


c  ^ 

Fig.  231. — Various  fleas,  magnified  about  30  diameters.  The  specimens  are 
treated  with  hot  20  per  cent,  caustic  potash  for  a  few  minutes,  dehydrated  in 
alcohol,  cleared  in  xylol  and  mounted  in  balsam,  a,  Ceratophyllus  fasciatus  d"; 
b,  Ceratophyllus  fasciatus,  9  \c ,  Leptopsylla  musculi,  cf ;  d,  Leptopsylla  musculi,  9 
(Bacot,  in  Journal  of  Hygiene,  "Plague  Supplement  in,  1914"). 

the  fleas  of  the  former  dog,  cat,  rat  or  mouse  tenants,  that  have  matured  or 
survived  the  interval  and  are  now  hungry  because  the  removal  of  the  family 
months  before,  was  probably  followed  by  the  withdrawal  of  the  rats  and  mice  no 
longer  able  to  find  food  in  the  deserted  habitation. 

To  get  rid  of  such  fleas  is  often  a  perplexing  question.  A  way  to  accomplish 
it  is  to  place  a  cage  containing  a  cat  or  a  guinea-pig,  or  a  trap  containing  living 
rats  or  mice  on  the  floor  of  a  room  and  surround  it  by  sticky  fly-paper.  Fleas 
when  empty  and  hungry,  were  found  by  Strickland*  to  be  able  to  jump  4  inches; 
those  recently  fed  only  3  inches.  In  their  endeavors  to  reach  the  caged  animals 
the  fleas  jump  upon  the  fly-paper  and  are  caught.  This  can  be  done  in  several 
rooms  of  the  house  and  soon  cleans  up  the  fleas. 

During  such  periods  of  fasting  the  sexes  do  not  copulate  and  no  ova  are  pro- 
duced^ As  soon  as  blood  is  taken,  copulation  takes  place,  and  if  the  blood  be 

*  "Journal  of  Hygiene,"  1914,  xiv,  p.  129. 


The  Plague  Fleas  561 

that  of  the  preferred  host,  ovulation  follows  in  about  twenty-four  hours.  The 
eggs  are  relatively  large,  and  small  numbers  are  produced. 

In  the  case  of  Sarcopsylla  penetrans,  a  flea  that  has  no  known  interest  in  con- 
nection with  plague  transmission,  the  female  after  copulation  imbeds  itself  in 
the  skin  of  the  host  and  suffers  an  enormous  saccular  distension  of  the  abdomen 
where  many  ova  are  produced.  Ordinary  fleas  never  imbed  themselves  but  sim- 
ply bite  and  suck  blood,  leaping  off  of  the  host  when  satisfied. 

Epidemics  of  plague  among  men  are  commonly  preceded  by  epizootics  of 
plague  among  rats.  The  mortality  of  the  rats  being  high  and  their  number 
diminishing,  many  fleas  are  unprovided  for  and  seek  human  hosts  upon  whom 
to  satisfy  their  appetites.  In  this  way,  the  plague  which  was  at  first  transmitted 
by  the  fleas  to  the  rats,  is  now  transmitted  to  men.  Human  fleas  may  also  trans- 


Fig.  232. — Various  fleas,  magnified  about  30  diameters.  The  specimens  are 
treated  with  hot  20  per  cent,  caustic  potash  for  a  few  minutes,  dehydrated  in 
alcohol,  cleared  in  xylol  and  mounted  in  balsam,  a,  Ctenocephalus  canis,  d" ;  b 
Ctenocephalus  canis,  9 ;  c,  Ctenocephalus  felis,  d" ;  d,  Ctenocephalus  f elis,  9 
(Bacot,  in  Journal  of  Hygiene,  "Plague  Supplement  in,  1914"). 


mit  the  infection  from  man  to  man,  but  the  bulk  of  the  transmission  probably 
takes  place  through  rat  fleas. 

When  the  plague  spreads  from  the  rat  to  ground  squirrels  or  to  marmots,  rare 
fleas  may  engage  in  the  transmission  of  the  disease  from  animal  to  animal  and 
from  man  to  man,  but  ordinarily  it  is  the  common  rat  fleas  that  are  responsible 
for  it. 

Both  rats  and  fleas  vary  in  prevalence  and  in  relative  frequency  in  different 
parts  of  the  world.  Thus  there  are  three  common  rats:  Mus  decumanus,  the 
brown  or  sewer  rat,  Mus  rattus,  the  black  or  house  rat  and  Mus  norvegius,  the 
Norway  rat.  In  Northern  Europe,  the  Mediterranean  coast,  Egypt  and  North 
America,  the  Norway  rat  has  colonized  more  or  less  successfully.  Where  it 
preponderates  Ceratophyllus  fasciatus  is  a  common  flea.  Where  Mus  decumanus 
and  Mus  rattus  alone  are  found,  or  are  preponderant,  Xenopsylla  cheopis  is  the 
common  flea.  In  the  Orient,  Xenopsylla  cheopis  is  the  chief  flea  that  is  to  be 
taken  into  account  in  plague  transmission.  The  dog  flea  Ctenocephalus  canis 
36 


562 


Plague 


is  common  everywhere  as  is  Pulex  irritans,  the  human  flea.  It  is  likely  that  any 
or  all  of  these  engage  in  plague  transmission  when  once  an  epidemic  has  started, 
but  the  most  active  vector  of  the  disease,  the  world  over,  and  the  most  important 
agent  in  starting  human  epidemics  of  plague  is  Xenopsylla  cheopis. 

Much  interesting  and  valuable  information  concerning  the  biology,  bionomics 


Fig.  233. — Various  fleas,  magnified  about  30  diameters.  The  specimens  are 
treated  with  hot  20  per  cent,  caustic  potash  for  a  few  minutes,  dehydrated  in 
alcohol,  cleared  in  xylol  and  mounted  in  balsam,  a,  Pulex  irritans,  cf ;  b,  Pulex 
irritans,  9  ;c,  Xenopsylla  cheopis,  cf ;  d,  Xenopsylla  cheopis,  9  (Bacot,  in  Journal  of 
Hygiene,  "Plague  Supplement  in,  1914"). 

and  relation  of  rats  and  fleas  to  plague,  will  be  found  in  the  "  Reports  of  the  India 
Plague  Commission "  many  of  which  are  to  be  found  in  the  "Journal  of  Hygiene," 
vols.  i-xiv. 

The  following  illustrations  and  tabulations  will  enable  the  student  to  identify 
the  common  genera  of  fleas.  For  more  intimate  systematic  study  he  must  be 
referred  to  "A  Text-book  of  Medical  Entomology,"  by  Patton  and  Cragg.* 

*  "  Christian  Literature  Society  of  India,  London,  Madras  and  Calcutta,"  1913. 


Table  for  Identification  of  Fleas 


563 


TABLE  FOR  THE  IDENTIFICATION  OF  THE  FLEAS  CONCERNED  IN 
PLAGUE  TRANSMISSION 

Family—  PULICID^. 
Subfamily— P  ULICIN&. 
All  have  eyes. 


A.  Have  no  combs  or  spines  on 
head,  thorax  or  abdomen. 


a.  The  meso-sternite  is  narrow 
and  has  no  rod-like  incrassa- 
tion  from  the  insertion  of  the 
coxa  upward Pulex. 


b.  The  meso-sternite  has  a  rod- 
like  incrassation  from  the 
insertion  of  the  coxa  up- 
ward   Xenopsylla. 


B.  With  combs. 


c.    Combs     on     the     prothorax 

only Ceratophyllus. 


d.  Combs  on  the  prothorax  and 
on  the  gena  or  lower  margin 
of  the  face Ctenocephalus. 


OTHER   MICRO-ORGANISMS   OF   THE   PLAGUE    GROUP 

The  Bacillus  pestis  is  a  member  of  a  group  of  organisms  col- 
lectively known  as  the  bacilli  of  hemorrhagic  septicemia.  Two  of 
these  organisms  are  of  sufficient  interest  to  deserve  special  mention. 


564  Micro-organisms  of  the  Plague  Group 

BACILLUS    CHOLERA    GALLINARUM    (PERRONCITO)  ;    BACILLUS 

CHOLERA;  BACILLUS  AVICIDUM;  BACILLUS  Avi- 

SEPTICUS;  BACILLUS  OF  RABBIT  SEPTI- 

CEMIA;  BACILLUS  CUNICULICIDA 

General  Characteristics. — A  non-motile,  non-flagellated,  non-sporogenous, 
non-liquefying,  non-chromogenic,  aerobic  bacillus,  pathogenic  for  birds  and 
mammals,  staining  by  the  ordinary  methods,  but  not  by  Gram's  nethod,  pro- 
ducing acids,  indol,  and  phenol,  and  coagulating  milk. 

The  barnyards  of  both  Europe  and  America  are  occasionally  visited  by  an 
epidemic  disease  known  as  "chicken-cholera,"  Huhnercholera,  or  cholera  de  poule, 
which  rapidly  destroys  pigeons,  turkeys,  chickens,  ducks,  and  geese.  Rabbit- 
warrens  are  also  at  times  affected  and  the  rabbits  killed. 

The  bacillus  responsible  for  this  disease  was  first  observed  by  Perroncito* 
in  1878,  and  afterward  thoroughly  studied  by  Toussaint  and  Pasteur. t 

Morphology. — The  organisms  are  short  and  broad,  with  rounded  ends,  measur- 
ing i  X  0.4  to  0.6  v,  sometimes  joined  to  produce  chains.  Pasteur  at  first 
regarded  them  as  diplococci,  because  the  poles  stain  intensely,  a  narrow  space 
between  them  remaining  almost  uncolored.  This  peculiarity  is  very  marked, 
and  careful  examination  is  required  to  detect  the  intermediate  substance.  The 
bacillus  does  not  form  spores,  is  not  motile,  and  has  no  flagella.  J 

Staining. — The  organism  stains  with  ordinary  anilin  dye  solutions,  but  not  by 
Gram's  method. 

Cultivation. — Colonies. — Colonies  upon  gelatin  plates  appear  after  about  two 
days  as  small,  irregular,  white  points.  The  deep  colonies  reach  the  surface  slow- 
ly, and  do  not  attain  to  any  considerable  size.  The  gelatin  is  not  liquefied.  The 
colonies  appear  under  the  microscope  as  irregularly  rounded  yellowish-brown 
disks  with  distinct  smooth  borders  and  granular  contents.  Sometimes  there  is  a 
distinct  concentric  arrangement. 

Gelatin. — In  gelatin  puncture  cultures  a  delicate  white  line  occurs  along  the 
entire  path  of  the  wire.  Upon  the  surface  the  development  is  much  more  marked, 
so  that  the  growth  resembles  a  nail  with  a  good-sized  flat  head.  If  the  bacilli  be 
planted  upon  the  surface  of  obliquely  solidified  gelatin,  a  much  more  pronounced 
growth  takes  place,  and  along  the  line  of  inoculation  a  dry,  granular  coating  is 
formed.  There  is  no  liquefaction  of  the  medium. 

Bouillon. — The  growth  in  bouillon  is  accompanied  by  a  slight  cloudiness. 

Agar. — This  growth,  like  that  upon  agar-agar  and  blood-serum,  is  white, 
shining,  rather  luxuriant,  and  devoid  of  characteristics. 

Potato. — Upon  potato  no  growth  occurs  except  at  37°C.  It  is  a  very  insig- 
nificant, yellowish-gray,  translucent  film. 

Milk  is  acidulated  and  slowly  coagulated. 

Vital  Resistance. — The  bacillus  readily  succumbs  to  the  action  of  heat  and 
dryness.  The  organism  is  an  obligatory  aerobe. 

Metabolic  Products. — Indol  and  phenol  are  formed.  Acids  are  produced  in 
sugar-containing  media,  without  gas  formation. 

Pathogenesis. — The  introduction  of  cultures  of  this  bacillus  into  chickens, 
geese,  pigeons,  sparrows,  mice,  and  rabbits  is  sufficient  to  produce  fatal  septice- 
mia.  Feeding  chickens,  pigeons,  and  rabbits  with  material  infected  with  the 
bacillus  is  also  sufficient  to  produce  the  disease.  Guinea-pigs,  cats,  and  dogs 
seem  immune,  though  they  may  succumb  to  large  doses  if  given  intraperitoneally. 
The  organism  is  probably  harmless  to  man. 

Fowls  ill  with  the  disease  fall  into  a  condition  of  weakness  and  apathy,  which 
causes  them  to  remain  quiet,  seemingly  almost  paralyzed,  and  the  feathers  ruffled 
up.  ^  The  eyes  are  closed  shortly  after  the  illness  begins,  and  the  birds  gradually 
fall  into  a  stupor,  from  which  they  do  not  awaken.  The  disease  is  fatal  in  from 
twenty-four  to  forty-eight  hours.  During  its  course  there  is  profuse  diarrhea, 
with  very  frequent  fluid,  slimy,  grayish-white  discharges. 

*  "  Archiv.  f.  wissenschaftliche  und  praktische  Thierheilkunde,"  1879. 

"  Compte-rendu  de  1'Acad.  de  Sci.  de  Paris,"  vol.  xc. 

j  Thoinot  and  Masselin  assert  that  the  organism  is  motile.  "Precis  de  Mi- 
crobie,"  2d  ed.,  1893. 


Chicken- Cholera  565 

Lesions. — The  autopsy  shows  that  when  the  bacilli  are  introduced  subcuta- 
neously  a  true  septicemia  results,  with  the  formation  of  a  hemorrhagic  exudate 
and  gelatinous  infiltration  at  the  seat  of  inoculation.  The  liver  and  spleen  are 
enlarged;  circumscribed,  hemorrhagic,  and  infiltrated  areas  occur  in  the  lungs; 
the  intestines  show  an  intense  inflammation  with  red  and  swollen  mucosa,  and 
occasional  ulcers  following  small  hemorrhages.  Pericarditis  is  frequent.  The 
bacilli  are  found  in  all  the  organs.  If,  on  the  other  hand,  the  disease  has  been 
produced  by  feeding,  the  bacilli  are  chiefly  to  be  found  in  the  intestine.  Pasteur 
found  that  when  pigeons  were  inoculated,  into  the  pectoral  muscles,  if  death  did 
not  come  on  rapidly,  portions  of  the  muscle  (sequestra)  underwent  degeneration 
and  appeared  anemic,  indurated,  and  of  a  yellowish  color. 

Immunity. — Pasteur*  discovered  that  when  cultures  are  allowed  to  remain 
undisturbed  for  several  months,  their  virulence  becomes  greatly  lessened,  and 
new  cultures  transplanted  from  them  are  also  attenuated.  If  chickens  be  inocu- 
lated with  such  attenuated  cultures,  no  other  change  occurs  than  a  local  inflam- 


Fig.  234. — Bacillus  of  chicken-cholera,  from  the  heart's  blood  of  a  pigeon. 
X  1000  (Frankel  and  Pfeiffef). 

matory  reaction  that  soon  disappears  and  leaves  the  birds  protected  against 
future  infection  with  virulent  bacilli.  From  these  observations  Pasteur  worked 
out  a  system  of  protective  vaccination  in  which  the  fowls  are  first  inoculated  with 
attenuated,  then  with  more  active,  and  finally  with  virulent,  cultures,  with  re- 
sulting protection  and  immunity. 

Use  has  been  made  of  this  bacillus  to  kill  rabbits  in  Australia,  where  they  are 
pests.  It  is  estimated  that  two  gallons  of  bouillon  culture  will  destroy  20,000 
rabbits,  irrespective  of  infection  by  contagion. 

The  bacillus  of  chicken-cholera  may  be  identical  with  organisms  found  in 
various  epidemic  diseases  of  larger  animals,  and,  indeed,  no  little  confusion  has 
arisen  from  the  description  of  what  is  now  pretty  generally  accepted  to  be  the 
same  organism  as  the  bacillus  of  rabbit  septicemia  (Koch),  Bacillus  cuniculicida 

*  An  interesting  account  of  Pasteur's  experiments  upon  chicken-cholera  can 
be  found  in  the  "Life  of  Pasteur,"  by  Vallery-Radot,  translated  by  Mrs.  R.  S. 
Devonshire,  1909.  Popular  Edition,  New  York,  Doubleday,  Page  and  Co. 


566  Micro-organisms  of  the  Plague  Group 

(Fliigge),  bacillus  of  "Wildseuche"  (Hiippe),  bacillus  of  "Biiffelseuche"  (Oriste- 
Armanni),  etc. 

BACILLUS  SUISEPTICUS  (LOFFLER  AND  SCHUTZ) 

General  Characteristics.— A  non-motile,  non-flagellated,  non-sporogenous, 
non-liquefying,  non-chromogenic,  aerobic  and  optionally  anaerobic  bacillus, 
pathogenic  for  hogs  and  many  other  animals,  staining  by  the  ordinary  methods, 
but  not  by  Gram's  method.  It  produces  a  slight  acidity  in  milk,  but  does  not 
coagulate  it. 

The  bacillus  of  swine-plague,  or  Bacillus- suisepticus  of  Loffler  and  Schiitz*  and 
Salmon  and  Smith,f  but  slightly  resembles  the  bacillus  of  hog-cholera  (q.v.), 
though  it  was  formerly  confounded  with  it  and  at  one  time  thought  to  be  iden- 
tical with  it.  The  species  have  sufficient  well-marked  characteristics,  however, 
to  make  their  differentiation  easy. 

Swine-plague  is  a  rather  common  and  exceedingly  fatal  epidemic  disease.  It 
not  infrequently  occurs  in  association  with  hog-cholera,  and  because  of  the  lack 
of  sufficiently  well-characterized  symptoms — sick  hogs  appearing  more  or  less 
alike — is  often  mistaken  for  it.  The  confusion  resulting  from  such  faulty 
diagnosis  makes  it  difficult  to  determine  exactly  how  fatal  either  may  be  in 
uncomplicated  cases. 

Morphology. — The  bacillus  of  swine-plague  much  resembles  that  of  chicken- 
cholera.  It  is  a  short  organism,  rather  more  slender  than  the  related  species, 
not  possessed  of  flagella,  incapable  of  movement,  and  producing  no  spores. 

It  is  an  optional  anaerobe. 

Staining. — The  bacillus  stains  by  the  ordinary  methods,  sometimes  only  at 
the  poles,  then  closely  resembling  the  bacillus  of  chicken-cholera.  It  is  not 
colored  by  Gram's  method. 

Cultivation. — In  general,  the  appearance  in  culture-media  is  very  similar  to 
that  of  the  hog-cholera  bacillus.  Kruse,J  however,  points  out  that  when  the 
bacillus  grows  in  bouillon  the  liquid  remains  clear,  the  bacteria  gathering  to 
form  a  flocculent,  stringy  sediment.  The  organism  does  not  grow  upon  ordi- 
nary acid  potato,  but  if  the  reaction  of  the  medium  be  alkaline,  a  grayish-yellow 
patch  is  formed.  In  milk  a  slight  acidity  is  produced,  but  the  milk  is  not 
coagulated. 

Vital  Resistance. — The  vitality  of  the  organism  is  low,  and  it  is  easily  destroyed. 
Salmon  says  that  it  soon  dies  in  water  or  when  dried,  and  that  the  temperature 
for  its  growth  must  be  more  constant  and  every  condition  of  life  more  favorable 
than  for  the  hog-cholera  bacillus.  The  organism  is  said  to  be  widely  distributed 
in  nature,  and  is  probably  present  in  every  herd  of  swine,  though  not  pathogenic 
except  when  its  virulence  becomes  increased  or  the  vital  resistance  of  the  animals 
diminished  by  some  unusual  condition. 

Rabbits,  mice,  and  small  birds  are  very  susceptible  to  the  infection,  usually 
dying  of  septicemia  in  twenty-four  hours;  guinea-pigs  are  less  susceptible,  except 
very  young  animals,  which  die  without  exception.  Chickens  are  more  immune, 
but  usually  succumb  to  large  doses.  Hogs  die  of  septicemia  after  subcutaneous 
injection  of  the  bacilli.  There  is  a  marked  edema  at  the  point  of  injection.  If 
injected  into  the  lung,  a  pleuropneumonia  follows,  with  multiple  necrotic  areas  in 
the  lung.  In  these  cases  the  spleen  is  not  much  swollen,  there  is  slight  gastro- 
intestinal catarrh,  and  the  bacilli  are  present  everywhere  in  the  blood. 

Animals  can  be  infected  only  by  subcutaneous,  intravenous,  and  intraperi- 
toneal  inoculation,  not  by  feeding. 

As  seen  in  hogs,  the  symptoms  of  swine-plague  closely  resemble  those  of  hog- 
cholera,  but  differ  in  the  occurrence  of  cough,  swine-plague  being  prone  to  affect 
the  lungs  and  oppress  the  breathing,  which  becomes  frequent,  labored,  and  pain- 
ful, while  hog-cholera  is  chiefly  characterized  by  intestinal  symptoms. 

The  course  of  the  disease  is  usually  rapid,  and  it  may  be  fatal  in  a  day  or  two. 

Lesions. — At  autopsy  the  lungs  are  found  to  be  inflamed,  and  to  contain 
numerous  small,  pale,  necrotic  areas,  and  sometimes  large  cheesy  masses  i  or 

*  "Arbeiten  aus  dem  kaiserlichen  Gesundheitsamte,"  I. 

t  "Zeitschrift  f.  Hygiene,"  x. 

JFlugge's  "Die  Mikroorganismen,  1896,"  p.  419. 


Swine-plague  567 

2  inches  in  diameter.  Inflammations  of  the  serous  membranes  affecting  the 
pleura,  pericardium,  and  peritoneum,  and  associated  with  fibrinous  inflammatory 
deposits  on  the  surfaces,  are  common.  There  may  be  congestion  of  the  mucous 
membrane  of  the  intestines,  particularly  of  the  large  intestine,  or  the  disease  in 
this  region  may  be  an  intense  croupous  inflammation  with  the  formation  of  a 
fibrinous  exudative  deposit  on  the  surface.  A  hemorrhagic  form  of  the  disease 
is  said  to  be  common  in  Europe,  but,  according  to  Salmon,  is  rare  in  the  United 
States. 


CHAPTER  XXVI 
ASIATIC  CHOLERA 

SPIRILLUM  CHOLERA  ASIATICS  (KOCH*) 

General  Characteristics. — A  motile,  flagellated,  non-sporogenous,  liquefying, 
non-chromogenic,  non-aerogenic,  parasitic  and  saprophytic,  pathogenic,  aerobic 
and  optionally  anaerobic  spirillum,  staining  by  ordinary  methods,  but  not  by 
Gram's  method. 

Cholera  is  a  disease  endemic  in  certain  parts  of  India  and  prob- 
ably indigenous  in  that  country.  Though  early  mention  of  it  was 
made  in  the  letters  of  travelers,  and  though  it  appeared  in  medical 
literature  and  in  governmental  statistics  more  than  a  century  ago, 
we  find  that  little  attention  was  paid  to  the  disease,  except  in  its 
disastrous  effect  upon  the  armies,  native  and  European,  of  India 
and  adjacent  countries.  The  opening  up  of  India  by  Great  Britain 
in  the  last  century  has  made  scientific  observation  of  the. disease 
possible  and  has  permitted  us  to  determine  the  relation  its  epidemics 
bear  to  the  manners  and  customs  of  the  people. 

The  filthy  habits  of  the  Oriental  people,  their  poverty,  crowded 
condition,  and  peculiar  religious  customs,  are  all  found  to  aid  in 
the  distribution  of  the  disease.  Thus,  the  city  of  Benares  drains 
into  the  Ganges  River  by  a  most  imperfect  system,  which  distributes 
the  greater  part  of  the  sewage  immediately  below  the  banks  upon 
which  the  city  is  built  and  along  which  are  the  numerous  "Ghats" 
or  staircases  by  which  the  people  reach  the  sacred  waters.  It  is 
a  matter  of  religious  observance  for  every  zealot  who  makes  a 
pilgrimage  to  the  "sacred  city"  to  take  a  bath  in  and  drink  a 
quantity  of  this  sacred  but  polluted  water,  and  il  may  be  imagined 
that  the  number  of  pious  Hindoos  who  leave  Benares  with  "comma 
bacilli"  in  their  intestines  or  upon  their  clothes  must  be  great,  for 
there  are  few  months  in  the  year  when  the  city  is  exempt  from 
the  disease. 

The  pilgrimages  and  great  festivals  of  both  Hindoos  and  Moslems, 
by  bringing  together  enormous  numbers  of  people  to  crowd  in  close 
quarters  where  filth  and  bad  diet  prevail,  cause  a  rapid  increase  in 
the  number  of  cases  during  these  periods  and  facilitate  the  distribu- 
tion of  the  disease  when  the  festivals  break  up.  Probably  no  more 
favorable  conditions  for  the  dissemination  of  a  disease  can  be  imagined 
than  occurs  with  the  return  of  the  Moslem  pilgrims  from  Mecca. 
The  disease  extends  readily  along  the  regular  lines  of  travel,  visiting 
town  after  town,  until  from  Asia  it  has  frequently  extended  into 

*  "Deutsche  med.  Wochenschrift,"  1884-1885,  Nos.  19,  20,  37,  38,  and  39. 

568 


Distribution 


569 


Europe,  and  by  steamships  plying  foreign  waters  has  several  times 
been  carried  to  our  own  continent.  Many  cases  are  on  record  which 
show  conclusively  how  a  single  ship,  having  a  few  cholera  cases  on 
board,  may  be  the  starting-point  of  an  outbreak  of  the  disease  in 
the  port  at  which  it  arrives. 

The  most  recent  great  epidemic  of  cholera  began  in  1883.  From 
Asia  it  spread  westward  throughout  Europe,  extended  by  means 
of  the  steamship  lines  to  numerous  of  the  large  ports,  of  which  Ham- 
burg in  Germany  suffered  most  acutely,  and  even  extended  to  some 
of  the  ports  of  Africa  and  America.  Russia  probably  suffered  more 
than  any  other  European  country,  and  it  is  estimated  that  in  that 
country  there  were  no  less  than  800,000  deaths.  During  1911  the 
disease  again  appeared  in  Europe  and  invaded  the  countries  along 
the  Mediterranean  coasts. 


Fig.  235. — Cholera  spirilla. 

Specific  Organism. — The  discovery  of  the  spirillum  of  cholera 
was  made  by  Koch  while  serving  as  a  member  of  a  German  com- 
mission appointed  to  study  the  disease  in  Egypt  and  India  in  1883-84. 
Since  its  discovery  the  spirillum  has  been  subjected  to  much  careful 
investigation,  and  an  immense  amount  of  literature,  a  large  part  of 
which  was  stimulated  by  the  Hamburg  epidemic  of  1892,  has 
accumulated. 

Distribution. — The  cholera  spirilla  can  be  found  with  great 
regularity  in  the  intestinal  evacuations  of  cholera  cases,  and  can 
often  be  found  in  drinking-water  and  milk,  and  upon  vegetables, 
etc.,  in  cholera-infected  districts.  There  can  be  little  doubt  that 
they  find  their  way  into  the  body  with  the  food  and  drink.  Cases 
in  the  literature  show  how  cholera  germs  enter  drinking-water  and 
are  thus  distributed;  how  they  are  sometimes  thoughtlessly  sprinkled 
over  green  vegetables  offered  for  sale  in  the  streets,  with  infected 


570  Asiatic  Cholera 

water  from  polluted  gutters;  how  they  enter  milk  with  water  used 
to  dilute  it;  how  they  appear  to  be  carried  about  in  clothing  and 
upon  food-stuffs;  how  they  can  be  brought  to  articles  of  food  by  flies 
that  have  preyed  upon  cholera  excrement;  and  other  interesting 
modes  of  infection.  The  literature  is  so  vast  that  it  is  scarcely 
possible  to  mention  even  the  most  instructive  examples.  A  bacteri- 
ologist became  infected  while  experimenting  with  the  cholera  spirilla 
in  Koch's  laboratory.  It  is  commonly  supposed  that  the  cholera 
organism  may  remain  alive  in  water  for  an  almost  unlimited  length 
of  time,  but  experiments  have  not  shown  this  to  be  the  case.  Thus, 
Wolffhiigel  and  Riedel  have  shown  that  if  the  spirilla  be  planted 
in  sterilized  water  they  grow  with  great  rapidity  after  a  short  time, 


Fig.  236. — Spirillum  of  Asiatic  cholera,  from  a  bouillon  culture  three  weeks  old, 
showing  long  spirals.     X  1000  (Frankel  and  Pfeiffer). 

and  can  be  found  alive  after  months  have  passed.^  Frankel,  how- 
ever, points  out  that  this  ability  to  grow  and  remain  vital  for  long 
periods  in  sterilized  water  does  not  guarantee  the  same  power  of 
growth  in  unsterilized  water,  for  in  the  latter  the  simultaneous 
growth  of  other  bacteria  serves  to  extinguish  the  cholera  spirilla 
in  a  few  days. 

Morphology. — The  micro-organism  described  by  Koch,  and 
now  generally  accepted  to  be  the  cause  of  cholera,  is  a  short  rod 
i  to  2  ju  in  length  and  0.5  /z  in  breadth,  with  rounded  ends,  and  a 
distinct  curve,  so  that  the  original  name  by  which  it  was  known,  the 
"comma  bacillus,"  applies  very  well.  One  of  the  most  common 
forms  is  that  in  which  two  short  curved  individuals  are  conjoined 
in  an  S-shape. 

When  the  conditions  of  nutrition  are  good,  multiplication  by  fission 
progresses  with  rapidity;  but  when  adverse  conditions  arise,  long 


Staining  571 

spiral  threads — unmistakable  spirilla — develop.  Frankel  found 
that  the  exposure  of  the  cultures  to  unusually  high  temperatures, 
the  addition  of  small  amounts  of  alcohol  to  the  culture-media,  and 
other  unfavorable  conditions  lead  to  the  production  of  spirals 
instead  of  "commas." 

The  cholera  spirilla  are  actively  motile,  and  in  hanging-drop 
preparations  can  be  seen  to  swim  about  with  great  rapidity.  Both 
comma-shaped  and  spiral  organisms  move  with  a  rapid  rotary 
motion. 

The  presence  of  a  single  flagellum  attached  to  one  end  can  be 
demonstrated  without  difficulty. 


.„-• 


Fig.  237. — Cover-glass  preparation  of  a   mucous   floccule   in    Asiatic   cholera. 

X  650  (Vierordt). 

Involution-forms  of  bizarre  appearance  are  common  in  old 
and  sometimes  in  fresh  cultures.  Many  individuals  show  by 
granular  cytoplasm  and  irregular  outline  that  they  are  degenerated. 
Cholera  spirilla  from  various  sources  differ  in  the  extent  of  involution. 

In  partially  degenerated  cultures  containing  long  spirals,  Hiippe 
observed,  by  examination  in  the  "  hanging-drop,"  certain  large 
spheric  bodies  which  he  described  as  spores  (arthrospores).  Koch 
and,  indeed,  all  other  observers  fail  to  find  spores  in  the  cholera 
organism,  and  the  nature  of  the  bodies  described  by  Hiippe  must 
be  regarded  as  doubtful. 

Staining. — The  cholera  spirillum  stains  well  with  the  ordinary 
aqueous  solutions  of  the  anilin  dyes,  especially  fuchsin.  At  times 
the  staining  must  be  continued  for  from  five  to  ten  minutes  to  se- 
cure homogeneity.  The  organism  does  not  stain  by  Gram's  method. 
It  may  be  colored  and  examined  while  alive;  thus,  Cornil  and 


572 


Asiatic  Cholera 


Babes,  in  demonstrating  it  in  the  rice-water  discharges,  "  spread  out 
one  of  the  white  mucous  fragments  upon  a  glass  slide  and  allow 
it  to  dry  partially;  a  small  quantity  of  an  exceedingly  weak  solu- 
tion of  methyl  violet  in  distilled  water  is  then  applied  to  it,  and  it  is 
flattened  out  by  pressing  down  a  cover-glass,  over  which  is  placed  a 
fragment  of  filter-paper,  which  absorbs  any  excess  of  fluid  at  the 
margin  of  the  cover-glass.  The  characteristics  of  comma  bacilli 
so  prepared  and  examined  with  an  oil-immersion  lens  (  X  700-800) 
are  readily  made  out  because,  though  they  take  up  enough  stain 
to  color  them,  they  still  retain  the  power  of  vigorous  movement, 
which  would  be  entirely  lost  if  the  specimen  were  dried,  stained,  and 
mounted  in  the  ordinary  fashion." 


Fig.  238. — Spirillum  of  Asiatic  cholera;  colonies  two  days  old  upon  a  gelatin 
plate.      X  35  (Heim). 

Isolation  of  the  Organism. — One  of  the  best  methods  of  securing 
a  pure  culture  of  the  cholera  spirillum,  and  also  of  making  a  bacterio- 
logic  diagnosis  of  the  disease  in  a  suspected  case,  is  probably  that 
of  Schottelius.  v 

A  small  quantity  of  the  fecal  matter  is  mixed  with  bouillon  and  stood  in  an 
incubating  oven  for  twenty-four  hours.  If  the  cholera  spirilla  are  present  they 
will  grow  most  rapidly  at  the  surface  of  the  liquid  where  the  supply  of  air  is  good. 
A  pellicle  will  be  formed,  a  drop  from  which,  diluted  in  melted  gelatin  and  poured 
upon  plates,  will  show  typical  colonies. 

Cultivation. — The  cholera  organism  is  easily  cultivated,  and 
grows  luxuriantly  upon  the  usual  laboratory  media. 

Colonies. — The  colonies  grown  upon  gelatin  plates  are  character- 
istic and  appear  in  the  lower  strata  of  the  gelatin  as  small  white 


Cultivation 


573 


dots,  which  gradually  grow  out  to  the  surface,  effect  a  slow  lique- 
faction of  the  medium,  and  then  appear  to  be  situated  in  little  pits 
with  sloping  sides.  The  appearance  suggests  that  the  plate  is 
full  of  little  holes  or  air-bubbles,  and  Is  due  to  the  slow  evaporation 
of  the  liquefied  gelatin. 

Under  the  microscope  the  colony  of  the  cholera  spirillum  is 
fairly  well  characterized.  The  little  colonies  that  have  not  yet 
reached  the  surface  of  the  gelatin  soon  show  a  pale  yellow  color  and 
an  irregular  contour.  They  are  coarsely  granular,  the  largest 
granules  being  in  the  center.  As  the  colony  increases  in  size  the 
granules  do  the  same  and  attain  a  peculiar  transparent  appearance 
suggestive  of  powdered  glass.  The  slow  liquefaction  causes  the 


Fig.  239. — Spirillum  cholerae  asiaticae;  gelatin  puncture  cultures  aged  forty-eight 
and  sixty  hours  (Shakespeare). 

colony  to  be  surrounded  by  a  transparent  halo.  As  the  liquefied 
gelatin  evaporates,  the  colony  begins  to  sink,  and  also  to  take  on  a 
peculiar  rosy  color. 

Gelatin. — In  puncture  cultures  in  gelatin  the  growth  is  also 
quite  characteristic.  It  occurs  along  the  entire  puncture,  but  best 
at  the  surface,  where  it  is  in  contact  with  the  atmosphere.  Lique- 
faction of  the  medium  begins  almost  at  once,  keeps  pace  with 
the  growth,  but  is  always  more  marked  at  the  surface  than  lower 
down.  The  result  is  the  formation  of  a  short,  rather  wide  funnel 
at  the  top  of  the  puncture.  As  the  growth  continues,  evapora- 
tion of  the  medium  takes  place  slowly,  so  that  the  liquefied  gelatin 
is  lower  than  the  surrounding  solid  portions,  and  the  growth  ap- 
pears to  be  surmounted  by  an  air-bubble. 


574  Asiatic  Cholera 

The  luxuriant  development  of  the  spirilla  in  the  liquefying  gelatin 
is  followed  by  the  formation  of  considerable  sediment  in  the  lower 
third  or  half  of  the  liquefied  area.  This  solid  material  consists  of 
masses  of  spirilla  which  have  probably  completed  their  life-cycle  and 
become  inactive.  Under  the  microscope  they  exhibit  the  most 
varied  involution-forms.  The  liquefaction  reaches  the  sides  of  the 
tube  in  from  five  to  seven  days,  but  is  not  complete  for  several 
weeks. 

Agar-agar. — When  planted  upon  the  surface  of  agar-agar  the 
spirilla  produce  a  grayish-white,  shining,  translucent  growth  along 
the  entire  line  of  inoculation.  It  is  in  no  way  peculiar  or  char- 
acteristic. The  vitality  of  the  organism  is  retained  much  better 
upon  agar-agar  than  upon  gelatin,  and,  according  to  Frankel,  the 
organism  can  be  transplanted  and  grown  when  nine  months  old. 

Blood-serum. — The  growth  upon  blood-serum  is  also  without 
distinct  peculiarities;  gradual  liquefaction  of  the  medium  occurs. 

Potato. — Upon  potato  the  spirilla  grow  well,  even  when  the 
reaction  is  acid.  In  the  incubator,  at  a  temperature  of  37°C.,  a 
transparent,  slightly  brownish  or  yellowish-brown  growth,  some- 
what resembling  that  of  glanders,  is  produced.  It  contains  large 
numbers  of  long  spirals. 

Bouillon. — In  bouillon  and  in  peptone  solution  the  cholera  organ- 
isms grow  well,  especially  upon  the  surface,  where  a  folded,  wrinkled 
pellicle  is  formed,  the  culture  fluid  remaining  clear. 

Milk. — In  milk  the  growth  is  luxuriant,  but  does  not  visibly 
alter  its  appearance.  The  existence  of  cholera  organisms  in  milk 
is,  however,  rather  short-lived,  for  the  occurrence  of  acidity  destroys 
them. 

Vital  Resistance. — Although  an  organism  that  multiplies  with 
great  rapidity  under  proper  conditions,  the  cholera  spirillum  does 
not  possess  much  resisting  power.  Sternberg  found  that  it  was 
killed  by  exposure  of  52°C.  for  four  minutes,  but  Kifasato  found  that 
ten  or  fifteen  minutes'  exposure  to  55°C.  was  not  always  fatal  to 
it.  In  a  moist  condition  the  organism  may  retain  its  vitality  for 
months,  but  it  is  very  quickly  destroyed  by  desiccation,  as  was 
found  by  Koch,  who  observed  that  when  dried  in  a  thin  film  its 
power  to  grow  disappeared  in  a  few  hours.  Kitasato  found  that 
upon  silk  threads  the  vitality  might  be  retained  longer.  Abel  and 
Claussen*  have  shown  that  it  does  not  live  longer  than  twenty  or 
thirty  days  in  fecal  matter,  and  often  disappears  in  from  one  to 
three  days.  The  organism  is  very  susceptible  to  the  influence  of 
carbolic  acid,  bichlorid  of  mercury,  and  other  germicides,  and  is 
also  destroyed  by  acids.  Hashimotof  found  that  it  could  not  live 
longer  than  fifteen  minutes  in  vinegar  containing  2.2-3.2  per  cent, 
of  acetic  acid. 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Tan.  31,  1895,  vol.  xvn,  No.  4. 
t  "Kwai  Med.  Jour.,"  Tokyo,  1893. 


Pathogenesis  575 

According  to  Frankel,  the  organisms  in  the  liquefied  cultures  all 
die  in  eight  weeks,  and  cannot  be  transplanted.  Kitasato,  how- 
ever, has  found  them  living  and  active  on  agar-agar  after  from 
ten  to  thirty  days,  and  Koch  occasionally  found  some  alive  after 
two  years. 

This  low  vital  resistance  of  the  microbe  is  very  fortunate,  for 
it  enables  us  to  establish  satisfactory  quarantine  for  the  prevention 
of  the  spread  of  the  disease.  Excreta,  soiled  clothing,  etc.,  are 
readily  rendered  harmless  by  the  proper  use  of  disinfectants.  Water 
and  food  are  rendered  innocuous  by  boiling  or  cooking.  Vessels 
may  be  disinfected  by  thorough  washing  with  jets  of  boiling  water 
discharged  through  a  hose  connected  with  a  boiler,  and  baggage 
can  be  sterilized  by  superheated  steam. 

Metabolic  Products. — Indol  is  one  of  the  characteristic  metabolic 
products  of  the  cholera  spirillum.  As  the  cholera  organisms  also 
produce  nitrites,  all  that  is  necessary  to  demonstrate  its  presence  in 
a  colorless  solution  is  to  add  a  drop  or  two  of  chemically  pure  sul- 
phuric acid,  when  the  well-known  reddish  color  will  appear. 

The  organism  also  produces  acid  in  milk  and  other  media.  Bitter 
has  also  shown  that  the  cholera  organism  produces  a  peptonizing 
and  probably  also  a  diastatic  ferment. 

Toxic  Products. — Rietsch  thinks  the  intestinal  changes  depend 
upon  the  action  of  the  peptonizing  ferment.  Cantani,  Nicati  and 
Rietsch,  Van  Ermengem,  Klebs,  and  others  found  toxic  effects 
from  cultures  administered  to  dogs  and  other  animals.  Several 
toxic  metabolic  products  of  the  spirilla  have  been  isolated.  Brieger,* 
Brieger  and  Frankel,  f  Gamaleia,f  Sobernheim,§  and  Villiers  have 
studied  more  or  less  similar  toxic  products.  The  real  toxic  sub- 
stance is,  however,  not  known. 

Pathogenesis. — Through  what  activity  the  cholera  organism 
provokes  its  pathogenic  action  is  not  yet  determined.  The  organ- 
isms, however,  abound  in  the  intestinal  contents,  penetrate  spar- 
ingly into  the  tissues,  but  slightly  invade  the  lymphatics,  and 
almost  never  enter  the  circulation;  hence  it  is  but  natural  to  conclude 
that  the  first  action  must  be  an  irritative  one  depending  upon  toxin- 
formation  in  the  intestine. 

In  the  beginning  of  the  disease  the  small  and  large  intestines 
are  deeply  congested,  almost  velvety  in  appearance,  and  contain 
liquid  fecal  matter.  The  patient  suffers  from  diarrhea,  by  which 
the  feces  are  hurried  on  and  become  extremely  thin  from  the  ad- 
mixture of  a  copious  watery  exudate.  As  the  feces  are  hurried  out, 
more  and  more  of  the  aqueous  exudate  accumulates,  until  the  intes- 
tine seems  to  contain  only  watery  fluid.  The  solitary  glands  and 
Peyer's  patches  are  found  enlarged  and  the  mucosa  becomes  macer- 

*  "Berliner  klin.  Wochenschrift,"  1887,  p.  817. 

"  Untersuchungen  iiber  die  Bakteriengifte,"  etc.,  Berlin,  1890. 
j  "Archiv  de  med.  exp.,"  rv,  No.  2. 
§  "Zeitschrift  fur  Hygiene,"  1893,  XIV>  I45- 


576  Asiatic  Cholera 

ated  and  necrotic,  its  epithelium  separating  in  small  shreds  or 
flakes.  The  evacuations  of  watery  exudate  rich  in  these  shreds  con- 
stitute the  characteristic  " rice- water  discharges"  of  the  disease. 
As  the  disease  progresses,  the  denudation  of  tissue  results  in  the 
formation  of  good-sized  ulcerations.  Perforations  and  deep  ulcer- 
ations  are  rare.  Pseudo-membranous  formations  not  infrequently 
occur  upon  the  abraded  and  ulcerated  surfaces.  The  other  mucous 
membranes  of  the  alimentary  apparatus  become  congested  and 
abraded;  the  parenchyma  of  the  liver,  kidneys,  and  other  organs 
become  markedly  degenerated,  so  that  the  urine  becomes  highly 
albuminous  and  very  scanty  in  consequence  of  the  anhydremia. 
The  cardio-vascular,  nervous,  and  respiratory  systems  present  no 
characteristic  changes. 

So  far  as  is  known,  cholera  is  a  disease  of  human  beings  only, 
and  never  occurs  spontaneously  in  the  lower  animals. 

Intraperitoneal  injection  of  the  virulent  cultures  produces  fatal 
peritonitis  in  guinea-pigs. 

Supposing  that  the  lower  animals  were  immune  against  cholera 
because  of  the  acidity  of  the  gastric  juice,  Nicati  and  Rietsch,* 
Van  Ermengem,  and  Kochj  have  suggested  methods  by  which 
the  micro-organisms  can  be  introduced  directly  into  the  intestine. 
The  first-named  investigatorsligated  the  common  bile-duct  of  guinea- 
pigs,  and  then  injected  the  spirilla  into  the  duodenum  with  a  hypo- 
dermic needle,  with  the  result  that  the  animals  usually  died,  some- 
times with  choleraic  symptoms.  The  excessively  grave  nature  of 
the  operation  upon  such  a  small  and  delicately  constituted  animal  as 
a  guinea-pig,  however,  greatly  lessens  the  value  of  the  experiment. 
Koch's  method  of  infection  by  the  mouth  is  much  more  satisfactory. 
By  injecting  laudanum  into  the  abdominal  cavity  of  guinea-pigs 
the  peristaltic  movements  of  the  intestine  can  be  checked.  The 
amount  necessary  for  the  purpose  is  large  and  amounts  to  about 
i  gram  for  each  200  grams  of  body-weight,.  It  completely  nar- 
cotizes the  animals  for  a  short  time  (one  to  two  hours),  but  they  re- 
cover without  injury.  The  contents  of  the  stomach  are  neutralized 
after  administering  the  opium,  by  introducing  5  cc.  of  a  5  per 
cent,  aqueous  solution  of  sodium  carbonate  through  a  pharyngeal 
catheter.  With  the  gastric  contents  thus  alkalinized  and  the  peris- 
talsis paralyzed,  a  bouillon  culture  of  the  cholera  spirillum  is  intro- 
duced through  the  stomach- tube.  The  animal  recovers  from  the 
manipulation,  but  shows  an  indisposition  to  eat,  is  soon  observed  to 
be  weak  in  the  posterior  extremities,  subsequently  is  paralyzed,  and 
dies  within  forty-eight  hours.  The  autopsy  shows  the  intestine 
congested  and  filled  with  a  watery  fluid  rich  in  spirilla — an  appear- 
ance which  Frankel  declares  to  be  exactly  that  of  cholera.  In 
man,  as  well  as  in  these  artificially  infected  animals,  the  spirilla  are 

*  "Deutsch.  med.  Wochenschrift,"  1884. 
t  Ibid.,  1885. 


Detection  of  the  Organism  577 

never  found  in  the  blood  or  tissues,  but  only  in  the  intestine,  where 
they  frequently  enter  between  the  basement  membrane  and  the 
epithelial  cells,  and  aid  in  the  detachment  of  the  latter. 

Issaeff  and  Kolle  found  that  when  virulent  cholera  spirilla  are 
injected  into  the  ear-veins  of  young  rabbits  the  animals  die  on  the 
following  day  with  symptoms  resembling  the  algid  state  of  human 
cholera.  The  autopsy  in  these  cases  showed  local  lesions  of  the 
small  intestine  very  similar  to  those  observed  in  cholera  in  man. 

Guinea-pigs  are  susceptible  to  intraperitoneal  injections  of 
the  spirillum,  and  speedily  succumb.  The  symptoms  are  rapid 
fall  of  temperature,  tenderness  over  the  abdomen,  and  collapse. 
The  autopsy  shows  an  abundant  fluid  exudate  containing  the  micro- 
organisms, and  injection  and  redness  of  the  peritoneum  and  viscera. 

Specificity. — The  cholera  spirillum  is  present  in  the  dejecta 
of  cholera  with  great  regularity,  and  as  regularly  absent  from  the 
dejecta  of  healthy  individuals  and  those  suffering  from  other  dis- 
eases. No  satisfactory  proof  of  the  specific  nature  of  the  organ- 
isms can  be  obtained  by  experimentation  upon  animals.  Ani- 
mals are  never  affected  by  any  disease  similar  to  cholera  during 
epidemics,  nor  do  foods  mixed  with  cholera  discharges  or  with  pure 
cultures  of  the  cholera  spirillum  affect  them.  Subcutaneous  in- 
oculations do  not  produce  cholera. 

Detection  of  the  Organism. — It  often  becomes  a  matter  of  im- 
portance to  detect  the  cholera  spirilla  in  drinking-water,  and,  as 
the  number  in  which  the  bacteria  exist  in  such  a  liquid  may  be 
very  small,  difficulty  may  be  experienced  in  finding  them  by  ordi- 
nary methods.  One  of  the  most  expeditious  methods  is  that 
recommended  by  Loffler,  who  adds  200  cc.  of  the  water  to  be 
examined  to  10  cc.  of  bouillon,  allows  the  mixture  to  stand  in  an 
incubator  for  from  twelve  to  twenty-four  hours,  and  then  makes 
plate  cultures  from  the  superficial  layer  of  the  liquid,  where,  if 
present,  the  development  of  the  spirilla  will  be  most  rapid  because 
of  the  free  access  of  air. 

Gordon*  employs  a  medium  composed  of  lemco  i  gram,  peptone 
i  gram,  sodium  bicarbonate  o.i  gram,  starch  i  gram,  and  distilled 
water  100  cc.  for  the  differentiation  of  the  cholera  and  Finkler- 
Prior  spirilla.  If  the  medium  be  tinted  with  litmus  and  the  cultures 
grown  at  37°C.,  a  strongly  acid  change  is  produced  by  the  true 
cholera  organism  in  twenty-four  hours.  The  Finkler-Prior  spirillum 
produces  but  slight  acidity,  which  first  appears  about  the  third 
day. 

The  identification  of  the  cholera  spirillum,  and  its  differen- 
tiation from  spiral  organisms  of  similar  morphology  obtained 
from  feces  or  water  in  which  no  cholera  organisms  are  expected,  is 
becoming  less  and  less  easy  as  our  knowledge  of  the  organisms 
increases.  The  following  points  may  be  taken  into  consideration: 

*  "British  Medical  Journal,"  July  28,  1906. 
37 


578  Asiatic  Cholera 

(i)  The  typical  morphology.  The  true  cholera  organism  is  short, 
has  a  single  curve,  is  rounded  at  the  ends,  and  possesses  a  single 
flagellum.  (2)  The  infectivity.  Freshly  isolated  cultures  should  be 
pathogenic  for  guinea-pigs  and  harmless  to  pigeons.  (3)  Vegeta- 
tive: The  organism  should  liquefy  10  per  cent,  gelatin  and  should 
not  coagulate  milk.  (4)  Metabolic:  the  indol  reaction  should  be 
marked.  (5)  Immunity  reactions:  the  organism  when  injected 
into  guinea-pigs  in  ascending  doses  should  occasion  immunity  against 
the  typical  cholera  organism,  and  the  serum  of  the  immunized 
guinea-pig,  when  introduced  into  a  new  guinea-pig,  should  protect 
it  from  infection  and  produce  Pfeiffer's  phenomenon.  The  blood- 
serum  of  animals  immunized  against  the  cholera  organism  should 
agglutinate  the  doubtful  organism  in  approximately  the  same 
dilution,  and  that  of  animals  immunized  to  the  doubtful  organism 
should  agglutinate  the  cholera  organism  reciprocally.  Both  organ- 
isms should  have  equal  capacity  for  absorbing  complements  and 
amboceptors  from  blood-serum.  (6)  The  true  cholera  organism 
should  not  be  hemolytic.  Too  much  reliance  must  not  be  placed 
upon  the  agglutination  tests  alone,  as  will  be  made  clear  by  a  perusal 
of  the  paper  upon  Bacteriological  Diagnosis  of  Cholera  by  Ruffer.* 

Pfeiffer  and  Vogedest  have  applied  the  "immunity  reaction" 
to  the  identification  of  cholera  spirilla  in  cultures.  A  hanging 
drop  of  a  i  :  50  mixture  of  a  powerful  anticholera  serum  and  a  par  dele 
of  cholera  culture  is  made  and  examined  under  the  microscope. 
The  cholera  spirilla  at  once  become  inactive,  and  are  in  a  short  time 
converted  into  little  rolled-up  masses.  If  the  culture  added  be  a 
spirillum  other  than  the  true  cholera  spirillum,  instead  of  being 
destroyed  the  micro-organisms  multiply  and  thrive  in  the  mixture 
of  serum  and  bouillon. 

Immunity. — One  attack  of  cholera  usually  leaves  the  victim 
immune  from  further  attacks  of  the  disease.  Gruber  and  Wiener,  f 
Haffkine,§  Pawlowsky,||  and  Pfeiffer**  have  immunized  animals 
against  toxic  substances  from  cholera  cultures  and  against  living 
cultures. 

Sobernheimf  f  found  the  Pfeiffer  reaction  specific  against  cholera 
alone,  and  thought  the  protection  not  due  to  the  strongly  bactericidal 
property  of  the  serum,  but  to  its  stimulating  effect  upon  the  body- 
cells;  for  if  the  serum  be  heated  to  6o°-7o°C.,  and  its  bactericidal 
power  thus  destroyed,  it  is  still  capable  of  producing  immunity. 
This,  of  course,  is  in  keeping  with  our  present  knowledge  of  the 
Immune  body,  which  is  not  destroyed  by  such  temperatures. 

*  "British  Medical  Journal,"  March  30,  1907,  i,  p.  735. 
t  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  March  21,  1896,  Bd.  xix,  No.  n. 
J  "Centralbi.  f.  Bakt.,"  1892,  xrv,  p.  76. 

§  "Le  Bull,  med.,"  1892,  p.  1113,  and  "Brit.  Med.  Jour.,"  1893,  p.  278. 
||  -'Deutsche  med.  Wochenschrift,"  1893,  No.  22. 
"  "Zeitschrift  fur  Hygiene,"  Bd.  xvm  and  xx. 
tt  "Zeitschrift  fur  Hygiene,  xx,  p.  438. 


Serum  Therapy  and  Prophylaxis  579 

The  immunity  produced  by  the  injection  of  the  spirilla  into 
guinea-pigs  continues  in  some  cases  as  long  as  four  and  a  half  months, 
but  the  power  of  the  serum  to  confer  immunity  is  lost  much  sooner. 

Serum  Therapy  and  Prophylaxis. — Of  the  numerous  attempts 
to  produce  immunity  against  cholera  in  man,  or  to  cure  cholera  when 
once  established  in  the  human  organism,  nothing  very  favorable  can 
be  said.  Experiments  in  this  field  are  not  new.  As  early  as  1885 
Ferran,  in  Spain,  administered  hypodermic  injections  of  pure 
virulent  cultures  of  the  cholera  spirillum,  in  the  hope  of  bringing 
about  immunity.  The  work  of  Haffkine,*  however,  is  the  chief 
important  contribution,  and  his  method  seems  to  be  followed  by  a 
positive  diminution  of  mortality  in  protected  individuals.  Haffkine 
uses  two  vaccines — one  mild,  the  other  so  virulent  that  it  would 
bring  about  extensive  tissue-necrosis  and  perhaps  death  if  used 
alone.  His  studies  embrace  more  than  40,000  inoculations  per- 
formed in  India.  The  following  extract  will  show  results  obtained 
in  1895: 

"  i.  In  all  those  instances  where  cholera  has  made  a  large  number  of  victims — 
that  is  to  say,  where  it  has  spread  sufficiently  to  make  it  probable  that  the  whole 
population,  inoculated  and  uninoculated,  were  equally  exposed  to  the  infection — 
in  all  these  places  the  results  appeared  favorable  to  inoculation. 

"2.  The  treatment  applied  after  an  epidemic  actually  breaks  out  tends  to 
reduce  the  mortality  even  during  the  time  which  is  claimed  for  producing  the 
full  effect  of  the  operation.  In  the  Goya  Garl,  where  weak  doses  of  a  relatively 
weak  vaccine  had  been  applied,  this  reduction  was  to  half  the  number  of  deaths; 
in  the  coolies  of  the  Assam-Burmah  survey  party,  where,  as  far  as  I  can  gather 
from  my  preliminary  information,  strong  doses  have  been  applied,  the  number 
of  deaths  was  reduced  to  one-seventh.  This  fact  would  justify  the  application 
of  the  method  independently  of  the  question  as  to  the  exact  length  of  time  during 
which  the  effect  of  this  vaccination  lasts. 

"3.  In  Lucknow,  where  the  experiment  was  made  on  small  doses  of  weak 
vaccines,  a  difference  in  cases  and  deaths  was  still  noticeable  in  favor  of  the 
inoculated  fourteen  to  fifteen  months  after  vaccination  in  an  epidemic  of  excep- 
tional virulence.  This  makes  it  probable  that  a  protective  effect  could  be 
obtained  even  for  long  periods  of  time  if  larger  doses  of  a  stronger  vaccine  were 
used. 

"4.  The  best  results  seem  to  be  obtained  from  application  of  middle  doses  of 
both  anticholera  vaccines,  the  second  one  being  kept  at  the  highest  possible 
degree  of  virulence  obtainable. 

"5.  The  most  prolonged  observations  on  the  effect  of  middle  doses  were  made 
in  Calcutta,  where  the  mortality  from  the  eleventh  up  to  the  four  hundred  and 
fifty-ninth  day  after  vaccination  was,  among  the  inoculated,  17.24  times  smaller, 
and  the  number  of  cases  19.27  times  smaller  than  among  the  not  inoculated." 

Pawlowsky  and  others  have  found  the  dog  susceptible  to  cholera, 
and  have  utilized  it  in  the  preparation  of  an  antitoxic  serum. 
The  dogs  were  first  immunized  against  attenuated  cultures,  then 
against  more  and  more  virulent  cultures,  until  a  serum  was  ob- 
tained whose  value  was  estimated  at  1:130,000  upon  experimental 
animals. 

Freymuthf    and    others    have    endeavored    to    secure   favorable 

*  "Le  Bull,  med.,"  1892,  p.  1113;  "Indian  Med.  Gazette,"  1893,  p.  97;  "Brit. 
Med.  Jour.,"  1893,  p.  278. 

t  "Deutsche  med.  Wochenschrift,"  1893,  No.  43. 


580  The  Finkler  and  Prior  Spirillum 

results  from  the  injection  of  blood-serum  from  convalescent  patients 
into  the  diseased.  One  recovery  out  of  three  cases  treated  is 
recorded. 

In  all  these  preliminaries  the  foreshadowing  of  a  future  thera- 
peusis  must  be  evident,  but  as  yet  nothing  satisfactory  has  been 
achieved. 

One  of  the  chief  errors  made  in  the  experimental  preparation  of 
anticholera  serums  is  that  efforts  have  been  directed  toward  endow- 
ing the  blood  with  the  power  of  resisting  and  destroying  the  bacteria 
that  rarely,  if  ever,  reach  it.  The  two  essentials  to  be  aimed  at  are 
an  antitoxin  to  neutralize  the  depressing  effects  of  the  toxalbumin, 
and  some  means  of  destroying  the  bacteria  in  the  intestine. 

Sanitation.- — The  first  appearance  of  cholera  may  depend  upon  the 
introduction  of  the  micro-organisms  upon  fomites,  hence  to  avoid 
epidemics  it  is  necessary  to  disinfect  all  such  coming  from  cholera- 
infected  localities. 

So  soon  as  cholera  asserts  itself,  the  chief  danger  lies  in  the  probable 
contamination  of  the  water-supply.  To  prevent  this  the  utmost 
effort  must  be  made  to  locate  all  cases  and  see  that  the  dejecta  are 
thoroughly  disinfected,  and  as  the  micro-organisms  persist  in  the 
intestinal  discharges  for  some  weeks  after  convalescence,  the  patients 
should  not  too  soon  be  discharged  from  the  hospital,  but  should 
be  retained  until  a  bacteriologic  examination  shows  no  more  comma 
bacilli  in  the  f  eces.  During  an  epidemic  the  water  consumed  should 
all  be  boiled,  raw  milk  should  be  avoided,  and  no  green  or  uncooked 
vegetables  or  fruits  eaten.  Foods  should  be  carefully  defended  from 
flies,  which  may  carry  the  organisms  to  them  and  infect  them. 
The  intestinal  evacuations  and  all  the  clothing,  bedding,  and  other 
articles  used  by  the  patients  should  be  carefully  disinfected. 

SPIRILLA  RESEMBLING  THE  CHOLERA  SPIRILLUM 
THE  FINKLER  AND  PRIOR  SPIRILLUM  (SPIRILLUM  PROTEUS) 

Similar  in  morphology  to  the  spirillum  of  cholera,  and  in  other  respects  closely 
related  to  it,  is  the  spirillum  obtained  from  the  f  eces  of  a  case  of  cholera  nostras 
by  Finkler  and  Prior.* 

Morphology. — It  is  shorter  and  stouter,  with  a  more  pronounced  curve  than 
the  cholera  spirillum,  and  rarely  forms  long  spirals.  The  central  portion  is  also 
somewhat  thinner  than  the  ends,  which  are  a  little  pointed  and  give  the  organism 
a  less  uniform  appearance.  Involution  forms  are  common  in  cultures,  and  appear 
as  spheres,  spindles,  clubs,  etc.  Like  the  cholera  spirillum,  each  organism  is 
provided  with  a  single  flagellum  situated  at  its  end,  and  is  actively  motile. 

Staining. — The  organism  stains  readily  with  the  ordinary  solutions,  but  not  by 
Gram's  method. 

Cultivation. — Colonies. — The  growth  upon  gelatin  plates  is  rapid,  and  leads  to 
such  extensive  liquefaction  that  four  or  five  dilutions  must  frequently  be  made  to 
secure  few  enough  organisms  to  enable  one  to  observe  the  growth  of  a  single 

*  "-Centralbl.  fur  allg.  Gesundheitspflege,"  Bonn,  1885,  Bd.  i;  "Deutsche 
med.  Wochenschrift,"  1884,  p.  632. 


Cultivation  581 

colony.  To  the  naked  eye  the  deep  colonies  appear  as  small  white  points.  They 
rapidly  reach  the  surface,  begin  liquefaction  of  the  gelatin,  and  by  the  second  day 
appear  about  the  size  of  lentils,  and  are  situated  in  little  depressions.  Under  the 


Fig.  240. — Spirillum  of  Finkler  and  Prior,  from  an  agar-agar  culture. 
(Itzerott  and  Niemann). 


X  1000 


microscope  they  are  yellowish  brown,  finely  granular,  and  are  surrounded  by 
a  zone  of  sharply  circumscribed  liquefied  gelatin.  Careful  examination  with  a 
high-power  lens  shows  rapid  movement  of  the  granules  in  the  colony. 


Fig.  241. — Spirillum  of  Finkler  and  Prior;  colony  twenty-four  hours  old,  upon  a 
gelatin  plate.      X  100  (Frankel  and  Pfeiffer). 

Gelatin  Punctures. — In  gelatin  punctures  the  growth  takes  place  rapidly  along 
the  whole  length  of  the  puncture,  forming  a  stocking-shaped  liquefaction  filled 
with  cloudy  fluid  which  does  not  precipitate  rapidly;  a  rather  smeary,  whitish 


582 


The  Finkler  and  Prior  Spirillum 


scum  is  usually  formed  upon  the  surface.  The  more  extensive  and  more  rapid 
liquefaction  of  the  medium,  the  wider  top  to  the  funnel,  the  absence  of  the  air- 
bubble,  and  the  clouded  nature  of  the  liquefied  material,  all  serve  to  differ- 
entiate the  culture  from  the  cholera  spirillum. 

Agar-agar.— Upon  agar-agar  the  growth  is  also  rapid,  and  in  a  short  time  th  e 
whole  surface  of  the  culture  medium  is  covered  with  a  moist,  thick,  slimy  coating, 
which  may  have  a  slightly  yellowish  tinge. 

Bouillon. — In  bouillon  the  organism  causes  a  diffuse  turbidity  with  a  more  or 
less  distinct  pellicle  on  the  surface.  In  sugar-containing  culture-media  it  causes 
no  fermentation  and  generates  no  gas. 

Potato. — The  cultures  upon  potato  are  also  different  from  those  of  the  cholera 
organism,  for  the  Finkler  and  Prior  spirilla  grow  rapidly  at  the  room  tempera- 
ture, and  produce  a  grayish-yellow,  slimy  shining  layer,  which  may  cover  the 
whole  of  the  culture-medium. 

Blood-serum. — Blood-serum  is  rapidly  liquefied  by  the  organism. 

Milk. — The  spirillum  does  not  grow  well  in  milk,  and  speedily  dies  in  water. 


Fig.  242. — Spirillum  of  Finkler  and  Prior;  gelatin  puncture  cultures  aged  forty- 
eight  and  sixty  hours  (Shakespeare). 

Metabolic  Products. — The  organism  does  not  produce  indol.  Buchner  has 
shown  that  in  media  containing  some  glucose  an  acid  reaction  is  produced.  Pro- 
teolytic  enzymes  capable  of  dissolving  gelatin,  blood-serum,  and  casein  are 
formed. 

Pathogenesis. — It  was  at  first  supposed  that  if  not  the  spirillum  of  cholera 
itself,  this  was  a  very  closely  allied  organism.  Later  it  was  supposed  to  be  the 
cause  of  cholera  nostras.  At  present  it  is  a  question  whether  the  organism  has 
any  pathologic  significance.  It  was  in  one  case  secured  by  Knisl  from  the  feces  of 
a  suicide,  and  has  been  found  in  carious  teeth  by  M  tiller. 

When  injected  into  the  stomach  of  guinea-pigs  treated  with  tincture  of  opium 
according  to  the  method  of  Koch,  about  30  per  cent,  of  the  animals  die,  but  the 
intestinal  lesions  produced  are  not  identical  with  those  produced  by  the  cholera 
spirillum.  The  intestines  in  such  cases  are  pale  and  filled  with  watery  material 
having  a  strong  putrefactive  odor.  This  fluid  teems  with  the  spirilla. 

It  seems  unlikely,  from  the  evidence  thus  far  collected,  that  the  Finkler  and 
Prior  spirillum  is  pathogenic  for  the  human  species.  As  Frankel  points  out,  it  is 
probably  a  frequent  and  harmless  inhabitant  of  the  human  intestine. 


Morphology 


583 


THE  SPIRILLUM  or  DENECKE  (SPIRILLUM  TYROGENUM) 

Another  organism  with  a  partial  resemblance  to  the  cholera  spirillum  was 
found  by  Denecke*  in  old  cheese. 


Fig.  243. — Spirillum  of  Denecke,  from  an  agar-agar  culture.      X  1000  (Itzerott 

and  Niemann). 

Morphology. — Its  form  is  similar  to  that  of  the  cholera  spirillum,  the  shorter 
individuals  being  of  equal  diameter  throughout.     The  spiral  forms  are  longer 


Fig.  244. — Spirillum  of  Denecke;  gelatin  puncture  cultures  aged  forty-eight  and 
sixty  hours  (Shakespeare). 

than  those  of  the  Finkler  and  Prior  spirillum,  and  are  more  tightly  coiled  than 
those  of  the  cholera  spirillum. 

*  "Deutsche  med.  Wochenschrift,"  1885. 


584  Spirillum  of  Gamaleia 

Like  its  related  species,  this  micro-organism  is  actively  motile  and  possesses  a 
terminal  flagellum. 

Cultivation. — It  grows  at  the  room  temperature,  as  well  as  at  37°C.,  in  this 
respect,  as  in  its  reaction  to  stains,  much  resembling  the  other  two. 

Colonies. — Upon  gelatin  plates  the  growth  of  the  colonies  is  much  more  rapid 
than  that  of  the  cholera  spirillum,  though  slower  than  that  of  the  Finkler  and 
Prior  spirillum.  The  colonies  appear  as  small  whitish,  round  points,  which  soon 
reach  the  surface  of  the  gelatin"  and  commence  liquefaction.  By  the  second 
day  each  is  about  the  size  of  a  pin's  head,  has  a  yellow  color,  and  occupies  the  bot- 
tom of  a  conical  depression.  The  appearance  is  much  like  that  of  colonies  of  the 
cholera  spirillum. 

The  microscope  shows  the  colonies  to  be  of  irregular  shape  and  coarsely  granu- 
lar, pale  yellow  at  the  edges,  gradually  becoming  intense  toward  the  center,  and 
at  first  circumscribed,  but  later  surrounded  by  clear  zones,  resulting  from  the 
liquefaction  of  the  gelatin.  These,  according  to  the  illumination,  appear  pale  or 
dark.  The  colonies  differ  from  those  of  cholera  in  the  prompt  liquefaction  of 
the  gelatin,  the  rapid  growth,  yellow  color,  irregular  form,  and  distinct  line  of 
circumscription. 

Gelatin  Punctures. — In  gelatin  punctures  the  growth  takes  place  all  along  the 
track  of  the  wire,  and  forms  a  cloudy  liquid  which  precipitates  at  the  apex  in  the 
form  of  a  coiled  mass.  Upon  the  surface  a  delicate,  imperfect,  yellowish  scum 
forms.  Liquefaction  of  the  entire  gelatin  generally  requires  about  two  weeks. 

Agar-agar. — Upon  agar-agar  this  spirillum  forms  a  thin  yellowish  layer  which 
spreads  quickly  over  most  of  the  surface. 

Bouillon. — In  bouillon  the  growth  of  the  organism  is  characterized  by  a  diffuse 
turbidity.  No  gas-formation  occurs  in  sugar-containing  media. 

Potatoes. — The  culture  upon  potato  is  luxuriant  if  grown  in  the  incubating 
oven.  It  appears  as  a  distinct  yellowish,  moist  film,  and  when  examined  micro- 
scopically is  seen  to  contain  beautiful  long  spirals. 

Metabolic  Products. — The  organism  produces  no  indol. 

Pathogenesis. — The  spirillum  of  Denecke  is  mentioned  only  because  of  its 
morphologic  resemblance  to  the  cholera  spirillum.  It  is  not  associated  with  any 
human  disease.  Experiments,  however,  have  shown  that  when  the  spirilla 
are  introduced  into  guinea-pigs  whose  gastric  contents  are  alkalinized  and  whose 
peristalsis  is  paralyzed  with  opium,  about  20  per  cent,  of  the  animals  die. 

THE  SPIRILLUM  OF  GAMALCIA*  (SPIRILLUM  METCHNIKOVI) 

Resembling  the  cholera  spirillum  in  morphology  and  vegetation,  and  possibly, 
as  has  been  suggested,  a  descendant  of  the  same  original  stock,  is  a  spirillum 
which  Gamaleia  cultivated  from  the  intestines  of  chickens  affected  with  a  disease 
similar  to  chicken-cholera. 

Morphology. — This  spirillum  is  a  trifle  shorter  and  thicker  than  the  cholera 
spirillum.  It  is  a  little  more  curved,  and  has.  similar  rounded  ends,  It  forms 
long  spirals  in  appropriate  media,  and  is  actively  motile.  Each  spirillum  is 
provided  with  a  terminal  flagellum.  No  spores  have  been  demonstrated. 

Staining. — The  organism  stains  easily,  the  ends  more  deeply  than  the  center. 
It  is  not  stained  by  Gram's  method. 

Cultivation, — It  grows  well  both  at  the  temperature  of  the  room  and  at  that  of 
incubation. 

Colonies. — The  colonies  upon  gelatin  plates  have  a  marked  resemblance  to 
those  of  the  cholera  spirillum,  yet  there  is  a  difference;  and  as  Pfeiffer  says,  "it  is 
comparatively  easy  to  differentiate  between  a  plate  of  pure  cholera  spirillum  and 
a  plate  of  pure  Spirillum  metchnikovi,  yet  it  is  almost  impossible  to  pick  out 
a  few  colonies  of  the  latter  if  mixed  upon  a  plate  with  the  former." 

Frankel  regards  this  organism  as  a  species  intermediate  between  the  cholera  and 
the  Finkler-Prior  spirillum. 

The  colonies  upon  gelatin  plates  appear  in  about  twelve  hours  as  small  whitish 
points,  and  rapidly  develop,  so  that  by  the  end  of  the  third  day  large  saucer-shaped 
liquefactions  resembling  colonies  of  the  Finkler-Prior  spirillum  occur.  Thelique- 
f  action -of  the  gelatin  is  quite  rapid,  the  resulting  fluid  being  turbid.  Usually, 
upon  a  plate  of  Vibrio  metchnikovi  some  colonies  are  present  which  closely 

*  "Ann.  de  PInst.  Pasteur,"  1888. 


Pathogenesis  585 

resemble  those  of  the  cholera  spirillum,  being  deeply  situated  in  conical  depres- 
sions in  the  gelatin.  Under  the  microscope  the  contents  of  the  colonies,  which 
appear  of  a  brownish  color,  are  observed  to  be  in  rapid  motion.  The  edges  of  the 
bacterial  mass  are  fringed  with  radiating  organisms. 

Gelatin  Punctures. — In  gelatin  tubes  the  growth  closely  resembles  that  of  the 
cholera  organism,  but  develops  more  slowly. 

Agar-agar. — Upon  the  surface  of  agar-agar  a  yellowish-brown  growth  develops 
along  the  whole  line  of  inoculation. 

Potato. — On  potato  at  the  room  temperature  no  growth  occurs,  but  at  the 
temperature  of  the  incubator  a  luxuriant  yellowish-brown  growth  takes  place. 
Sometimes  the  color  is  quite  dark,  and  chocolate-colored  potato  cultures  are  not 
uncommon. 

Bouillon. — In  bouillon  the  growth  which  occurs  at  the  temperature  of  the  incu- 
bator is  quite  characteristic,  and  very  different  from  that  of  the  cholera  spirillum. 
The  entire  medium  becomes  clouded,  of  a  grayish-white  color,  and  opaque.  A 
folded  and  wrinkled  pellicle  forms  upon  the  surface. 

Milk.— When  grown  in  litmus  milk,  the  original  blue  color  is  changed  to  pink  in 
a  day,  and  at  the  end  of  another  day  the  color  is  all  destroyed  and  the  milk  coagu- 


^£P^ 


Fig.  245. — Spirillum  metchnikovi,  from  an  agar-agar  culture.     X  1000  (Itzerott 

and  Niemann). 

lated.  Ultimately  the  clots  of  casein  sediment  in  irregular  masses,  from  the 
clear,  colorless  whey. 

Vital  Resistance. — The  organism,  like  the  cholera  vibrio,  is  very  susceptible  to 
the  influence  of  acids,  high  temperatures,  and  drying.  The  thermal  death-point 
is  5o°C.,  continued  for  five  minutes. 

Metabolic  Products. — The  addition  of  sulphuric  acid  to  a  culture  grown  in  a 
medium  rich  in  peptone  produces  the  same  rose  color  observed  in  cholera  cultures 
and  shows  the  presence  of  indol.  When  glucose  is  added  to  the  bouillon  no  fer- 
mentation or  gas-production  results.  The  organism  produces  acids  and  curdling 
enzymes. 

Pathogenesis. — The  organism  is  pathogenic  for  animals,  but  not  for  man. 
Pf  eiffer  has  shown  that  chickens  and  guinea-pigs  are  highly  susceptible,  and  when 
inoculated  under  the  skin  usually  die.  The  virulent  organism  is  invariably  fatal 
for  pigeons.  W.  Rindfleisch  has  pointed  out  that  this  constant  fatality  for 
pigeons  is  a  valuable  criterion  for  the  differentiation  of  this  spirillum  from  that 
of  cholera,  as  the  subcutaneous  injection  of  the  most  virulent  cholera  cultures  is 
never  fatal  to  pigeons,  the  birds  only  dying  when  the  injections  are  made  into  the 
muscles  in  such  a  manner  that  the  muscular  tissue  is  injured  and  becomes  a  locus 
minoris  resistentia.  When  guinea-pigs  are  treated  by  Koch's  method  of 
narcotization  and  cholera  infection,  the  temperature  of  the  animal  rises  for  a 
short  time,  then  abruptly  falls  to  33°C.  or  less.  Death  follows  in  from  twenty  to 


586  Spirillum  Schuylkiliensis 

twenty-four  hours.  A  distinct  inflammation  of  the  intestine,  with  exudate  and 
numerous  spirilla,  may  be  found.  The  spirilla  can  also  be  found  in  the  heart's 
blood  and  in  the  organs  of  such  guinea-pigs.  When  the  bacilli  are  introduced 
by  subcutaneous  inoculation,  the  autopsy  shows  a  bloody  edema  and  a  superficial 
necrosis  of  the  tissues. 

The  organisms  can  be  found  in  the  blood  and  all  the  organs  of  pigeons  and 
young  chickens,  in  such  large  numbers  that  Pfeiffer  has  called  the  disease  Vibrio- 
nensepticcemia.  In  the  intestines  very  few  alterations  are  noticeable,  and  very 
few  spirilla  can  be  found. 

Immunity. — Gamaleia  has  shown  that  pigeons  and  guinea-pigs  can  be  made 
immune  by  inoculating  them  with  cultures  sterilized  for  a  time  at  a  temperature 
of  ioo°C.  Mice  and  rabbits  are  immune,  except  to  very  large  doses. 


Fig.  246. — Spirillum  metchnikovi;  puncture  culture  in  gelatin  forty-eight  hours 
old  (Frankel  and  Pfeiffer). 

SPIRILLUM  SCHUYLKILIENSIS  (ABBOTT) 

Morphology. — This  micro-organism,  closely  resembling  the  cholera  spirillum, 
was  found  by  Abbott*  in  sewage-polluted  water  from  the  Schuylkill  River  at 
Philadelphia. 

Cultivation. — Colonies. — The  colonies  developed  upon  gelatin  plates  very 
closely  resemble  those  of  the  Spirillum  metschnikovi. 

Gelatin  punctures. — In  gelatin  puncture  cultures  the  appearance  is  exactly  like 
the  true  cholera  spirillum.  At  times  the  growth  is  a  little  more  rapid. 

Agar-agar. — The  growth  on  agar  is  luxuriant,  and  gives  off  a  pronounced  odor 
of  indol. 

Blood-serum. — Loffler's  blood-serum  is  apparently  not  a  perfectly  adapted 
medium,  but  upon  it  the  organisms  grow,  with  resulting  liquefaction. 

Potato. — Upon  potato,  at  the  point  of  inoculation  a  thin,  glazed,  more  or  less 
dirty  yellow  growth,  shading  to  brown  and  sometimes  surrounded  by  a  flat,  dry, 
lusterless  zone,  is  formed. 

Milk. — In  litmus  milk  a  reddish  tinge  develops  after  the  milk  is  kept  twenty- 
four  hours  at  body  temperature.  After  forty-eight  hours  this  color  is  increased 
and  the  milk  coagulates. 

Metabolic  Products. — In  peptone  solutions  indol  is  easily  detected.  No  gas  is 
produced  in  glucose-containing  culture-media.  Acids  and  coagulating  enzymes 
are  formed.  The  organism  is  a  facultative  anaerobe. 

*  "Journal  of  Experimental  Medicine,"  July,  1896,  vol.  i,  No.  3,  p.  419. 


Spirillum  Schuylkiliensis  587 

Vital  Resistance. — The  thermal  death  point  is  50°  C.  maintained  for  five 
minutes. 

Pathogenesis. — The  organism  is  pathogenic  for  pigeons,  guinea-pigs,  and  mice, 
behaving  much  like  Spirillum  metchnikoyi.  No  Pfeiffer's  phenomenon  was 
observed  with  the  use  of  serum  from  immunized  animals. 

Immunity. — Immunity  could  be  produced  in  pigeons,  and  it  was  found  that  the 
serum  was  protective  against  both  Spirillum  schuylkiliensis  and  Spirillum  metch- 
nikovi,  the  immunity  thus  produced  being  of  about  ten  days'  duration. 

In  a  second  paper  by  Abbott  and  Bergey*  it  was  shown  that  the  spirilla 
occurred  in  the  water  during  all  four  seasons  of  the  year,  and  in  all  parts  of  the 
river  within  the  city,  both  at  low  and  at  high  tide.  They  were  also  found  in  the 
sewage  emptying  into  the  river,  and  in  the  water  of  the  Delaware  River  as  fre- 
quently as  in  that  of  the  Schuylkill. 

One  hundred  and  ten  pure  cultures  were  isolated  from  the  sources  mentioned 
and  subjected  to  routine  tests.  It  was  found  that  few  or  none  of  them  were  iden- 
tical in  all  points.  There  seems  to  be,  therefore,  a  family  of  river  spirilla,  closely 
related  to  one  another,  like  the  different  colon  bacilli. 

The  opinion  expressed  is  that  "  the  only  trustworthy  difference  between  many 
of  these  varieties  and  the  true  cholera  spirillum  is  the  specific  reaction  with  serum 
from  animals  immune  against  cholera,  or  by  Pfeiffer's  method  of  intraperitoneal 
testing  in  such  animals." 

In  discussing  these  spirilla  of  the  Philadelphia  water  Bergey f  says: 

"The  most  important  point  with  regard  to  the  occurrence  of  these  organisms 
in  the  river  water  around  Philadelphia  is  the  fact  that  similar  organisms  have  been 
found  in  the  surface  waters  of  the  European  cities  in  which  there  had  recently 
been  an  epidemic  of  Asiatic  cholera,  notably  at  Hamburg  and  Altona.  .  .  . 
The  foremost  bacteriologists  of  Europe  have  been  inclined  to  the  opinion  that  the 
organisms  which  they  found  in  the  surface  waters  of  the  European  cities  were  the 
remains  of  the  true  cholera  organism,  and  that  the  deviations  in  the  morphologic 
and  biologic  characters  from  those  of  the  cholera  organism  were  brought  about 
by  their  prolonged  existence  in  water.  No  such  explanation  of  the  occurrence 
of  the  organisms  in  Philadelphia  waters  can  be  given." 

A  number  of  interesting  spirilla,  more  or  less  closely  resembling  that  of  Asiatic 
cholera,  have  been  described  from  time  to  time.  Their  variation  from  the  true 
cholera  organism  can  best  be  determined  by  an  examination  of  the  following 
table,  though  for  precise  information  the  student  will  do  well  to  look  up  the 
original  descriptions,  references  to  which  are  given  in  each  case. 

*  "Journal  of  Experimental  Medicine,"  vol.  n,  No.  5,  p.  535. 
t  "Journal  Amer.  Med.  Assoc.,"  Oct.  23,  1897. 


588 


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DIFFERENTIAL  T 

• 

Intestinal  Group. 

Spirillum  cholerae  asiaticae  (Koch*) 
Spirillum  cholerae  nostras  (Spit 
(Finkler  and  Priorf)  .  . 
Spirillum  tyrogenum  (Denecke  j) 
Spirillum  metschnikovi  (Gamaleia  \ 

Water  Group. 

Spirillum  dunbarensis  (Dimbar  ||) 
Spirillum  danubicus  (Heider**)  . 
Spirillum  I  (Wernicke-fi)  .... 
Spirillum  II  (Wernicke  it)  .... 
Spirillum  liquefaciens  (Bonhoffgg) 
Spirillum  weibeli  (Weibel  ||||)  .  .  . 
Spirillum  milleri  (Miller***)  .  .  . 
Spirillum  terrigenus  (Giintherttt) 
Spirillum  berolmensis  (Neisser  ttt) 
Spirillum  aquatilis  (Giintherggg)  . 
Spirillum  schuylkiliensis  (Abbott  an 

*  "  Berliner  klin.  Wochenschrift,"  i 
2  "  Ann.  de  1'Inst.  Pasteur,"  1888, 
ft"  Archiv  fiir  Hygiene,"  xxi,  1894, 
ill  "  Centralbl.  f.  Bakt.,"  etc.,  Bd  n, 
|U"  Hygienische  Rundschau,"  1893 

CHAPTER  XXVII 
TYPHOID  FEVER 

BACILLUS  TYPHOSUS  (EBERTH-GAFFKY) 

General  Characteristics. — A  motile,  flagellated,  non-sporogenous,  non- 
liquefying,  non-chromogenic,  non-aerogenic,  aerobic  and  optionally  anaerobic, 
pathogenic  bacillus,  staining  by  ordinary  methods,  but  not  by  Gram's  method, 
not  forming  indol,  acids  from  sugars,  or  coagulating  milk. 

Typhoid  fever,  "typhus  abdominalis,"  enteric  fever,  "la  fievre 
typhique,"  is  a  disease  so  well  known  and  of  such  universal  distribu- 
tion, that  no  introductory  remarks  concerning  it  are  necessary. 

The  bacillus  of  typhoid  fever  (Bacillus  typhosus)  was  discovered 


Fig.  247. — Bacillus  typhosus,  from  twenty-four-hour  culture  on  agar.     (From 
Hiss  and  Zinsser,  "Text-book  of  Bacteriology,"  D.  Appleton  &  Co.,  publishers.) 

in  1880  by  Eberth*  and  Koch,f  and  was  first  secured  in  pure  culture 
from  the  spleen  and  lymphatic  glands  four  years  later  by  Gaffky.J 
Distribution. — The  bacillus  is  both  saprophytic  and  parasitic. 
It  finds  abundant  opportunity  in  nature  for  growth  and  devel- 
opment, and,  enjoying  strong  resisting  powers,  can  accommodate 
itself  to  its  environment  much  better  than  the  majority  of  pathogenic 
bacteria,  and  can  be  found  in  water,  soiled  clothing,  dust,  sew- 
age, milk,  etc.,  contaminated  directly  or  indirectly  with  the  intestinal 
discharges  of  diseased  persons. 

*  "Virchow's  Archiv,"  1881  and  1883. 

t  "  Mittheilungen  aus  dem  kaiserl.  Gesundheitsamte,"  i,  45. 

j  Ibid.,  2. 

589 


590 


Typhoid  Fever 


Morphology. — The  typhoid  bacillus  measures  about  i  to  3  /*  (2  to 
4  n — Chantemesse,  Widal)  in  length  and  0.5  to  0.8  ^  in  breadth 
(Sternberg).  The  ends  are  rounded,  and  it  is  exceptional  for  the 
bacilli  to  be  united  in  chains.  The  size  and  morphology  vary  with 
the  nature  of  the  culture-medium  and  the  age  of  the  culture.  Thoi- 
not  and  Masselin,*  in  describing  these  morphologic  variations,  point 
out  that  when  grown  in  bouillon  the  typhoid  bacillus  is  very  slender; 
in  milk  it  is  stouter;  upon  agar-agar  and  potato  it  is  thick  and  short; 
and  in  old  gelatin  cultures  it  forms  long  filaments.  It  produces 
no  spores. 

Flagella.— The  organisms  are  actively  motile  and  are  provided 
with  numerous  flagella,  which  arise  from  all  parts  of  the  bacillus 
(peritricha),  and  are  10  to  20  in  number.  They  stain  well  by 


Fig.  248. — Bacillus  typhosus. 

Loffier's  method.  The  movements  of  the  short  bacilli  are  oscillating; 
those  of  the  longer  bacilli,  serpentine  and  undulating. 

Staining. — The  organism  stains  quite  well  by  the  ordinary  methods, 
but  not  by  Gram's  method.  As  it  gives  up  its  color  in  the  presence 
of  almost  any  solvent,  it  is  difficult  to  stain  in  tissue. 

When  sections  of  tissue  are  to  be  stained  for  the  demonstration  of 
the  typhoid  bacilli,  the  best  method  is  to  allow  them  to  remain  in 
Loffler's  alkaline  methylene  blue  for  from  fifteen  minutes  to  twenty- 
four  hours,  then  wash  in  water,  dehydrate  rapidly  in  alcohol,  clear 
up  in  xylol,  and  mount  in  Canada  balsam.  Ziehl's  method  also 
gives  good  results:  The  sections  are  stained  for  fifteen  minutes  in  a 
solution  of  distilled  water,  100,  fuchsin  i,  and  phenol  5.  After 
staining  they  are  washed  in  distilled  water  containing  i  per  cent. of 
acetic  acid,  dehydrated  in  alcohol,  cleared,  and  mounted.  In  such 
*  "Precis  de  Microbie,"  Paris,  1893. 


Cultivation  591 

preparations  the  bacilli  are  always  found  in  scattered  groups,  which 
are  easily  discovered,  under  a  low  power  of  the  microscope,  as 
reddish  specks,  and  readily  resolved  into  bacilli  with  the  oil-im- 
mersion lens. 

In  bacilli  stained  with  the  alkaline  methylene-blue  solution, 
dark-colored  dots  (Babes-Ernst  or  metachromatic  granules)  may 
sometimes  be  observed  near  the  ends  of  the  rods. 

Isolation. — The  bacillus  can  be  secured  in  pure  culture  from  an 
enlarged  lymphatic  gland  or  from  the  splenic  pulp  of  a  case  of  typhoid. 

As  the  groups  of  bacilli  are  sometimes  widely  scattered  through- 
out the  spleen,  E.  Frankel  recommends  that  as  soon  as  the  organ 
is  removed  from  the  body  it  be  wrapped  in  cloths  wet  with  a  solution 
of  bichlorid  of  mercury  and  kept  for  three  days  in  a  warm  room,  in 
order  that  a  considerable  and  massive  development  of  the  bacilli 


Fig.  249. — Bacillus  typhi  abdominalis ;  superficial  colony  two  days  old,  as  seen 
upon  the  surface  of  a  gelatin  plate.      X  20  (Heim). 

may  take  place.  The  surface  is  then  seared  with  a  hot  iron  and  ma- 
terial for  cultures  obtained  by  introducing  a  platinum  loop  into  the 
substance  of  the  organ  through  the  sterilized  surface. 

Cultures  may  be  more  easily  obtained  from  the  blood  of  the 
living  patients.  (See  "  Blood  culture,"  under  the  section  "Bacterio- 
logic  Diagnosis.") 

The  bacilli  can  also  be  secured  from  the  alvine  discharges  of 
typhoid  patients  during  the  second  and  third  weeks  of  the  disease. 

Cultivation. — The  bacillus  grows  well  upon  all  culture-media 
under  both  aerobic  and  anaerobic  conditions. 

Colonies. — The  deep  colonies  upon  gelatin  plates  appear  under  the 
microscope  of  a  brownish-yellow  color  and  spindle-shape,  and  are 
sharply  circumscribed.  When  superficial,  however,  they  become 
larger  and  form  a  thin,  bluish,  iridescent  layer  with  notched  edges. 
The  superficial  colonies  are  often  described  as  resembling  grapevine 
leaves  in  shape.  The  center  of  the  superficial  colonies  is  the  only 


592  Typhoid  Fever 

portion  which  shows  the  yellowish-brown  color.  The  gelatin  is  not 
liquefied. 

Gelatin  Punctures. — When  transferred  to  gelatin  puncture  cul- 
tures, the  typhoid  bacilli  develop  along  the  entire  track  of  the  wire, 
with  the  formation  of  minute,  confluent,  spheric  colonies.  A  small, 
thin,  whitish  layer  develops  upon  the  surface  near  the  center.  The 
gelatin  is  not  liquefied,  but  is  sometimes  slightly  clouded  in  the  neigh- 
borhood of  the  growth. 

Agar-agar. — The  growth  upon  the  surface  of  obliquely  solidified 
gelatin,  agar-agar,  or  blood-serum  is  not  luxuriant.  It  forms  a  thin, 
moist,  shining,  translucent  band  with  smooth  edges  and  a  grayish- 
yellow  color. 

Potato. — When  potato  is  inoculated  and  stood  in  the  incubating 
oven,  no  growth  can  be  seen  even  at  the  end  of  the  second  day, 
but  if  the  surface  of  the  medium  be  touched  with  a  platinum  wire, 
it  is  found  entirely  covered  with  a  rather  thick,  invisible  layer  of 
sticky  vegetation  which  the  microscope  shows  to  be  made  up  of 
bacilli.  This  is  described  as  the  invisible  growth.  Unfortunately, 
it  is  not  a  constant  characteristic,  for  occasionally  a  typhoid  bacillus 
will  show  a  distinct  yellowish  or  brownish  color.  The  typical 
growth  seems  to  take  place  only  when  the  reaction  of  the  potato  is 
acid. 

Bouillon. — In  bouillon  the  only  change  produced  by  the  growth  of 
the  bacillus  is  a  diffuse  cloudiness.  Rarely  a  pellicle  is  formed.  When 
sugars  are  added  to  the  bouillon  the  typhoid  bacillus  is  found  to 
form  acid  from  dextrose,  levulose,  galactose,  mannite,  maltose,  and 
dextrin,  but  not  to  form  acid  from  lactose  or  saccharose.  No  gas 
is  formed  in  the  fermentation  tube  with  any  of  the  sugars.  No  indol 
is  formed. 

Milk. — In  milk  containing  litmus  a  very  slight  and  slow  acidity 
is  produced,  which  later  gives  place  to  distinct  alkalinity.  The 
milk  is  not  coagulated. 

Vital  Resistance. — The  organisms  grow  well  at  all  ordinary  tem- 
peratures. The  thermal  death-point  is  given  by  Sternberg  at  56°C., 
destruction  being  effected  in  ten  minutes.  Upon  ordinary  culture- 
media,  the  organisms  remain  alive  for  several  months  if  drying  is 
prevented.  In  carefully  sealed  agar-agar  tubes  Hiss  found  the  or- 
ganism still  living  after  thirteen  years.'  According  to  Klemperer  and 
Levy,*  the  bacilli  can  remain  vital  for  three  months  in  distilled  water, 
though  in  ordinary  water  the  commoner  and  more  vigorous  sapro- 
phytes outgrow  them  and  cause  their  disappearance  in  a  few  days. 
There  seems  to  be  some  doubt,  however,  on  this  point,  as  Tavelf 
found  that  it  lived  for  six  months  in  the  blind  terminal  of  a  water- 
supply  pipe,  and  Hofmann,t  after  planting  it  in  an  aquarium  con- 

*  "-Clinical  Bacteriology."  Translated  by  A.  A.  Eshner,  Phila.,  W.  B.  Saun- 
ders  Co.,  1900. 

"Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1903,  xxxm,  p.  166. 
j  "Archiv.  f.  Hyg.,"  1905,  LII,  2,  208. 


Toxic  Products  593 

taining  fish,  snails,  water-plants,  and  protozoa,  was  able  to  recover 
it  from  the  water  after  thirty-six  days,  and  from  the  mud  in  the  bot- 
tom after  two  months.  In  elaborate  experimental  studies  of  this 
question  Jordan,  Russel,  and  Zeit*  found  its  longevity  to  be  only 
three  or  four  days  under  conditions  resembling  as  nearly  as  possible 
those  found  in  nature.  When  buried  in  the  upper  layers  of  the  soil 
the  bacilli  retain  their  vitality  for  nearly  six  months.  Robertsonf 
found  that  when  planted  in  soil  and  occasionally  fed  by  pouring 
bouillon  upon  the  surface,  the  typhoid  bacillus  maintained  its  vitality 
for  twelve  months.  He  suggests  that  it  may  do  the  same  in  the  soil 
about  leaky  drains. 

Cold  has  little  effect  upon  typhoid  bacilli,  for  some  can  withstand 
freezing  and  thawing  several  times.  Observing  that  epidemics  of 
typhoid  fever  had  never  been  traced  to  polluted  ice,  Sedgwick  and 
Winslowf  made  some  investigations  to  determine  what  quantitative 
reduction  might  be  brought  about  by  freezing,  and  accordingly  ex- 
perimentally froze  a  large  number  of  samples  of  water  intentionally 
infected  with  large  numbers  of  typhoid  bacilli  from  different  sources. 
It  was  found  that  the  bacilli  disappeared  in  proportion  to  the  length 
of  time  the  water  was  frozen,  and  that  the  reduction  averaged  99  per 
cent,  in  two  weeks.  The  last  two  or  three  bacilli  per  thousand 
appeared  very  resistant  and  sometimes  remained  alive  after  twelve 
weeks. 

They  have  been  found  to  remain  alive  upon  linen  from  sixty  to 
seventy-two  days,  and  upon  buckskin  from  eighty  to  eighty-five 
days. 

The  typhoid  bacillus  resists  the  action  of  chemic  agents  rather 
better  than  most  non-sporogenous  organisms.  The  addition  of 
from  o.i  to  0.2  per  cent,  of  carbolic  acid  to  the  culture-media  is 
without  effect  upon  its  growth.  At  one  time  the  tolerance  to  carbolic 
acid  was  thought  to  be  characteristic,  but  it  is  now  known  to  be  shared 
by  other  bacteria  (colon  bacillus).  It  is  killed  by  i  :  500  bichlorid 
of  mercury  solutions  and  5  per  cent,  carbolic  acid  solutions  in  five 
minutes. 

Metabolic  Products. — The  typhoid  bacillus  does  not  produce  indol. 
It  produces  a  small  amount  of  acid  when  grown  in  sugar-containing 
media,  but  its  regular  tendency  is  to  form  alkalies,  as  is  shown  by  the 
reactions  in  litmus  milk.  It  forms  no  coagulating  or  proteolytic 
enzymes. 

Toxic  Products. — The  disproportion  of  local  to  constitutional  dis- 
turbance in  typhoid  fever  and  the  irritative  and  necrotic  charac- 
ter of  its  lesions  suggest  that  we  have  to  do  with  a  toxic  organism. 
Brieger  and  Frankel  have,  indeed,  separated  a  toxalbumin,  which 
they  thought  to  be  the  specific  poison,  from  bouillon  cultures.  When 

*  "Journal  of  Infectious  Diseases,"  1904,  I,  p.  641. 

"Brit.  Med.  Jour.,"  Jan.  8,  1898. 
j  "Jour.  Boston  Soc.  of  Med.  Sci.,"  March  20, 1900,  vol.  iv,  No.  7,  p.  181. 

38 


594  Typhoid  Fever 

injected  into  guinea-pigs  the  typhotoxin  of  Brieger  causes  salivation, 
accelerated  respiration,  diarrhea,  mydriasis,  and  death  in  from 
twenty-four  to  forty-eight  hours.  Klemperer  and  Levy  also  point 
out,  as  affording  clinical  proof  of  the  presence  of  toxin,  the  occasional 
fatal  cases  in  which  the  typical  picture  of  typhoid  has  been  without 
the  characteristic  postmortem  lesions,  the  diagnosis  being  made  by 
the  discovery  of  the  bacilli  in  the  spleen. 

Pfeiffer  and  Kolle*  found  toxic  substance  in  the  bodies  of  the 
bacilli  only.  It  was  not,  like  the  toxins  of  diphtheria  and  tetanus, 
dissolved  in  the  culture-medium.  This  was  an  obstacle  to  the  immu- 
nization experiments  of  both  Pfeiffer  and  Kolle  andLoffler  and  Abel,| 
for  the  only  method  of  immunizing  animals  was  to  make  massive 
agar-agar  cultures,  scrape  the  bacilli  from  the  surface,  and  distribute 
them  through  an  indifferent  fluid  before  injecting  them  into  animals. 

If  the  bacilli  grown  upon  ordinary  culture-media  are  several  times 
washed  in  distilled  water,  and  then  allowed  to  macerate  in  normal 
salt  solution,  autolysis  takes  place  and  a  toxin  is  liberated,  showing 
that  the  toxin  is  intraceliular.  Macfadyen  and  Rowland  {liberated 
an  intraceliular  toxin  from  cultures  of  the  typhoid  bacilli  by  freezing 
them  with  liquid  air  and  grinding  them  in  an  agate  mortar.  Animals 
immunized  with  this  poison  produced  an  antiserum  active  against  it, 
but  useless  against  infection  with  typhoid  bacilli.  Wright,  of  Net- 
ley  §,  observes  that  Macfadyen's  method  of  securing  this  intracel- 
iular toxin  was  unnecessarily  cumbersome,  as  the  body  juices  of 
animals  injected  with  dead  cultures  of  the  bacilli  dissolve  them  at 
once  and  thus  liberate  the  same  toxic  product. 

Besredka||  and  Macfadyen**  think  that  exotoxin  is  also  formed. 
Vaughanff  has  obtained  poisonous  and  non-poisonous  fractions  by 
extracting  massive  cultures  of  typhoid  bacilli  with  2  per  cent,  solu- 
tions of  sodium  hydrate  in  absolute  alcohol  at  78°C. 

Mode  of  Infection. — The  typhoid  bacillus  enters  the  body  by 
way  of  the  alimentary  tract  with  infected  foods  and  water. 

Rosenau,  Lumsden,  and  Kastletf  were  able  to  connect  10  per  cent, 
of  the  cases  of  typhoid  fever  occurring  in  the  District  of  Columbia 
with  infection  through  milk.  Interesting  additional  facts  upon  the 
subject  can  be  found  in  Bulletin  No.  41  of  the  Hygienic  Laboratory 
upon  "Milk  in  its  Relation  to  the  Public  Health."  The  bacillus 
occasionally  enters  milk  in  water  used  to  dilute  it  or  to  wash  the  cans. 

The  occurrence  of  typhoid  fever  among  the  soldiers  of  the  United 
States  Army  during  the  Spanish- American  War  in  1898  was  shown  by 

*  "Deutsche  med.  Wochenschrift,"  Nov.  12,  1896. 

t  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Jan.  23,  1896,  Bd.  xix,  No.  23,  p.  51. 

i"Brit.  Med.  Jour.,"  1903. 

§  Ibid.,  April,  4,  1903,  i,  p.  786. 

||  "Ann.  de  i'lnst.  Pasteur,"  1895,  x,  1896,  xi. 

**  "Centralbl.  f.  Bakt.,"  etc.,  1906,  i. 

'  "Amer.  Jour.  Med.  Sci.,"  1908,  cxxxvi. 
JJ  "Hygienic  Laboratory  Bulletin  No.  33,"  Washington,  D.  C.,  1907. 


Pathogenesis  595 

Reed,  Vaughan,  and  Shakespeare*  to  be  largely  the  result  of  the 
pollution  of  the  food  of  the  soldiers  by  flies  that  shortly  before  had 
visited  infected  latrines. 

The  bacillus  is  also  occasionally  present  upon  green  vegetables 
grown  in  soil  fertilized  with  infected  human  excrement  or  sprinkled 
with  polluted  water,  and  epidemics  are  reported  in  which  the  occur- 
rence of  the  disease  was  traced  to  oysters  infected  through  sewage. 
Newsholmef  found  that  in  56  cases  of  typhoid  fever  about  one-third 
were  attributable  to  eating  raw  shell-fish  from  sewage-polluted  beds. 

Pathogenesis. — The  primary  activities  of  the  typhoid  bacillus 
are  unknown.  It  is  supposed  that  it  passes  uninjured  through  the 
acid  secretions  of  the  stomach  to  enter  the  intestine,  where  local  dis- 
turbances are  set  up.  Whether  during  an  early  residence  in  the 
intestine  its  metabolism  is  accompanied  by  the  formation  of  a  toxic 
product,  irritating  to  the  mucosa,  and  affording  the  bacilli  means  of 
entrance  to  the  lymph-vessels,  through  diminutive  breaches  of  con- 
tinuity, is  not  known.  We  usually  find  it  well  established  in  the 
intestinal  and  mesenteric  lymphatics  at  the  time  we  are  able  to 
recognize  the  disease. 

It  is  quite  certain  that  the  chief  operations  of  the  typhoid 
bacillus  are  in  the  tissues  and  not  in  the  intestine,  as  seems  to  be  a 
widely  prevalent  error.  It  is  contrary  to  most  of  our  knowledge  of 
the  organism  that  it  should  easily  adapt  itself  to  saprophytic  exist- 
ence among  the  more  vigorous  intestinal  organisms.  Those  who 
look  for  it  in  the  feces  are  usually  surprised  at  the  difficulty  of  finding 
it,  or  at  the  small  numbers  present.  It  is  far  more  easy  to  isolate 
the  organism  from  the  blood  than  from  the  feces,  and  much  greater 
numbers  occur  in  the  urine  than  in  the  feces.  It  probably  es- 
capes from  the  blood  into  the  bile,  where  it  grows  luxuriantly, 
and  entering  the  gall-bladder  may  take  up  permanent  residence 
there,  escaping  into  the  intestine  each  time  the  gall-bladder  is 
emptied.  Many  bacilli  thus  discharged  probably  meet  with  destruc- 
tion in  the  intestine,  though  some  convalescents  from  typhoid  fever 
for  years  have  a  periodic  appearance  of  bacilli  in  the  feces.  Such 
individuals  have  become  known  as  "  typhoid  carriers  "  and  are  a  men- 
ace to  the  public. 

In  a  case  studied  by  Miller  {  bacilli  were  found  in  the  gall-bladder 
seven  years  after  recovery  from  typhoid  fever;  in  a  case  studied  by 
Droba§  they  were  found  in  both  the  gall-bladder  and  a  gall-stone 
seventeen  years  after  recovery  from  the  disease;  Humer,||  found 
them  in  the  gall-bladder  of  a  patient  suffering  from  cholecystitis, 
eighteen  years  after  recovery  from  an  attack  of  typhoid  fever,  and  in  a 

*  "Report  on  Typhoid  Fever  in  the  U.  S.  Military  Camps  in  the  Spanish  War," 
vol.  i. 

t  "Brit.  Med.  Jour.,"  Jan.,  1895. 
j  "Bull,  of  the  Johns  Hopkins  Hospital,"  May,  1898. 
'Wiener  klin.  Wochenschrift,"  1809,  X1I>  P-  1141. 
Bull,  of  the  Johns  Hopkins  Hospital,"  Aug.  and  Sept.,  1899. 


596  Typhoid  Fever 

case  studied  by  Dean,*  they  were  present  in  the  stools  of  a  man 
twenty-nine  years  after  he  had  had  an  attack  of  typhoid  fever. 
Cushingt  invariably  found  the  bacilli  in  the  bile  in  clumps 
resembling  the  agglutinations  of  the  Widal  reaction.  He  thinks 
it  probable  that  these  dumps  form  nuclei  upon  which  bile  salts 
can  be  precipitated  and  calculous  formation  begun.  The  presence 
of  gall-stones,  together  with  the  long-lived  infective  agents,  may 
at  any  subsequent  time  provoke  cholecystitis.  Gushing  collected 
6  cases  of  operation  for  cholecystitis  with  calculi  in  which  typhoid 
bacilli  were  present,  and  5  in  which  Bacillus  coli  was  present 
in  the  gall-bladder. 


Fig.  250.— Intestinal  perforation  in  typhoid  fever.  Observe  the  threads  of 
tissue  obstructing  the  opening.  (Museum  of  the  Pennsylvania  Hospital.; 
(Keen,  "Surgical  Complications  and  Sequels  of  Typhoid  Fever.") 

With  the  most  approved  methods  yet  devised,  Peabody  and 
Pratt  J  were  unable  to  recover  the  micro-organism  from  the  intestinal 
contents  in  more  than  21  per  cent,  of  febrile  cases,  and  only  in  small 
numbers  as  a  rule.  The  greatest  number  was  obtained  when  there 
was  much  blood  in  the  stooL 

There  is  always  well-marked  blood-infection  during  the  first 
weeks  of  the  disease,  and  upon  this  depends  the  occurrence  of  the 
rose-colored  spots. 

British  Medical  Journal,''  March  7,  1008,  i,  p.  562. 

BuHof  the  Johns  Hopkins  Hospital,"  tt.  No. 86. 

Journal  of  the  American  Medical  Association,"  Sept  7, 1907,  fflx,  p.  846. 


*  .. 


.. 


Pathogenesis  597 

The  bacilli  enter  the  solitary  glands  and  Peyer's  patches,  and 
multiply  slowly  during  the  incubation  period  of  the  disease — one  to 
three  weeks.  The  immediate  result  of  their  activity  in  the  lymphatic 
structures  is  an  increase  in  the  number  of  cells,  the  ultimate  effect 
is  necrosis  and  sloughing  of  the  Peyer's  patches  and  solitary  glands. 
From  the  intestinal  lymphatics  the  bacilli  pass,  in  all  probability, 
to  the  mesenteric  nodes,  which  become  enlarged,  softened,  and 
sometimes  rupture.  They  also  invade  the  spleen,  liver  and  some- 
times the  kidneys,  and  other  organs  where  they  may  be  found 
in  small  clusters  in  properly  stained  specimens. 

Mallory*  found  the  histologic  lesions  of  typhoid  fever  to  be  wide- 
spread throughout  the  body  and  not  limited  to  the  Peyer's  patches  of 
the  intestine,  where  they  are  most  evident.  His  conclusions  regard- 
ing the  pathology  of  the  disease  are  briefly:  "The  typhoid  bacillus 
produces  a  mild  diffusible  toxin,  partly  within  the  intestinal  tract, 
partly  within  the  blood  and  organs  of  the  body.  This  toxin  pro- 
duces proliferation  of  the  endothelial  cells,  which  acquire  for  a 
certain  length  of  time  malignant  properties.  The  new-formed  cells 
are  epithelioid  in  character,  have  irregular,  lightly  staining,  ec- 
centrically situated  nuclei,  abundant,  sharply  denned,  acidophilic 
protoplasm,  and  are  characterized  by  marked  phagocytic  properties. 
These  phagocytic  cells  are  produced  most  abundantly  along  the  line 
of  absorption  from  the  intestinal  tract,  both  in  the  lymphatic  ap- 
paratus and  in  the  blood-vessels.  They  are  also  produced  by  dis- 
tribution of  the  toxin  through  the  general  circulation,  in  greatest 
numbers  where  the  circulation  is  slowest.  Finally,  they  are  pro- 
duced all  over  the  body  in  the  lymphatic  spaces  and  vessels  by 
absorption  of  the  toxin  eliminated  from  vthe  blood-vessels.  The 
swelling  of  the  intestinal  lymphoid  tissue  of  the  mesenteric  lymph 
nodes  and  of  the  spleen  is  due  almost  entirely  to  the  forma- 
tion of  phagocytic  cells.  The  necrosis  of  the 'intestinal  lymphoid 
tissue  is  accidental  in  nature  and  is  caused  through  occlusion 
of  the  veins  and  capillaries  by  fibrinous  thrombi,  which  owe 
their  origin  to  degeneration  of  phagocytic  cells  beneath  the 
lining  endothelium  of  the  vessels.  Two  varieties  of  focal  lesions 
occur  in  the  liver:  one  consists  of  the  formation  of  phagocytic  cells 
in  the  lymph-spaces  and  vessels  around  the  portal  vessels  under 
the  action  of  the  toxin  absorbed  by  the  lymphatics;  the  other  is 
due  to  obstruction  of  liver  capillaries  by  phagocytic  cells  derived 
in  small  part  from  the  lining  endothelium  of  the  liver  capillaries, 
but  chiefly  by  embolism  through  the  portal  circulation  of  cells 
originating  from  the  endothelium  of  the  blood-vessels  of  the  in- 
testine and  spleen.  The  liver-cells  lying  between  the  occluded 
capillaries  undergo  necrosis  and  disappear.  Later  the  foci  of  cells 
degenerate  and  fibrin  forms  between  them.  Invasion  by  poly- 
morphonuclear  leukocytes  is  rare." 

*  "Journal  of  Experimental  Medicine,"  1898,  vol.  in,  p.  611. 


598  Typhoid  Fever 

".  .  .  Histologically  the  typhoid  process  is  proliferative  and 
stands  in  close  relationship  to  tuberculosis,  but  the  lesions  are  diffuse 
and  bear  no  intimate  relation  to  the  typhoid  bacillus,  while  the 
tubercular  process  is  focal  and  stands  in  the  closest  relation  to  the 
tubercle  bacillus." 

The  growth  of  the  bacilli  in  the  kidneys  causes  albuminuria,  and  the 
bacilli  can  be  found  in  the  urine  in  about  25  per  cent,  of  the  cases. 
Smith*  found  them  in  the  urine  in  3  out  of  7  cases  which  he  investi- 
gated; Richardson,  f  in  9  out  of  38  cases.  They  did  not  occur  before 
the  third  week,  and  remained  in  one  case  twenty-two  days  after 
cessation  of  the  fever.  Sometimes  they  were  present  in  immense 
numbers,  the  urine  being  actually  clouded  by  their  presence. 
Petruschkyt  found  that  albuminuria  sometimes  occurs  without 
the  presence  of  the  bacilli;  that  their  presence  in  the  urine  is  infre- 
quent; that  the  bacilli  never  appear  in  the  urine  in  the  early  part  of 
the  disease,  and  hence  are  of  little  importance  for  diagnostic  pur- 
poses. Gwyn§  has  found  as  many  as  50,000,000  typhoid  bacilli 
per  cubic  centimeter  of  urine,  and  mentions  a  case  of  Cushing's  in 
which  the  bacilli  persisted  in  the  urine  for  six  years  after  the  primary 
attack  of  typhoid  fever.  Their  occurrence,  no  doubt,  depends  pri- 
marily upon  a  typhoid  bacteremia,  by  which  they  are  brought  to  the 
kidney.  Their  persistence  in  the  urine  after  recovery  from  typhoid 
fever,  depends  upon  continued  growth  in  the  bladder  and  not  upon 
continuous  escape  from  the  blood.  It  is  of  importance  from  a 
sanitary  point  of  view  to  remember  that  the  urine  as  well  as  the  feces 
is  infectious. 

The  bacilli  pass  from  the  lymphatics  to  the  general  circulation,  so 
that  all  cases  of  typhoid  fever  are  true  bacteremias  during  part  or  all 
of  their  course. 

Bacilli  can  be  found  in  the  circulating  blood.  The  eruption 
may  be  regarded  as  one  of  the  4ocal  irritative  manifestations  of  the 
bacillus,  as  the  blood  from  the  roseolas  always  contains  them,  and 
Richardson 1 1  found  it  necessary  to  examine  a  number  of  spots  in 
each  case.  He  carefully  disinfected  the  skin,  freezing  it  with 
chlorid  of  ethyl,  making  a  crucial  incision,  and  cultivating  from 
the  blood  thus  obtained.  He  was  able  to  secure  the  typhoid 
bacillus  in  13  out  of  14  cases  examined. 

As  a  means  of  diagnosis  the  matter  is  of  some  importance,  as  the 
rose  spots  may  precede  the  occurrence  of  the  Widal  reaction  by  a 
number  of  days. 

In  rare  instances  the  bacillus  may  be  found  in  the  expectoration, 
especially  when  pulmonary  complications  arise  in  the  course  of  the 

*  "Brit.  Med.  Jour.,"  Feb.  13,  1897. 

t  "Journal  of  Experimental  Medicine,"  May,  1898. 

"  Centralbl.  f.  Bakt.  u.  Parasitenk.,"  May  13,  1898,  No.  13,  p.  577. 
§  "Phila.  Med.  Jour.,"  March  3,  1900. 
II  "Phila.  Med.  Jour.,"  March  3,  1900. 


Prophylaxis  599 

disease.  Cases  of  this  kind  have  been  reported  by  Chantemesse  and 
Widal*  and  Frankel.f 

The  pyogenic  power  of  the  typhoid  bacillus  was  first  pointed 
out  by  A.  Frankel,  who  observed  it  in  a  suppuration  that  occurred 
four  months  after  convalescence.  Lowf  found  virulent  typhoid 
bacilli  in  the  pus  of  abscesses  occurring  from  one  to  six  years  after 
convalescence. 

Weichselbaum  has  seen  general  peritonitis  from  rupture  of  the 
spleen  in  typhoid  fever,  with  escape  of  the  bacilli.  Otitis  media, 
ostitis,  periostitis,  and  osteomyelitis  are  common  results  of  the 
lodgment  of  the  bacilli  in  bony  tissue.  Ohlmacher§  has  found  the 
bacilli  in  suppurations  of  the  membranes  of  the  brain.  The  bacilli 
are  also  encountered  in  other  local  suppurations  occurring  in  or 
following  typhoid  fever.  Flexner  and  Harris  ||  have  seen  a  case  in 
which  the  distribution  of  the  bacilli  was  sufficiently  widespread  to 
constitute  a  real  septicemia. 

Lower  Animals. — Typhoid  fever  is  communicable  to  animals  with 
difficulty.  They  are  not  infected  by  bacilli  contained  in  fecal  matter 
or  by  the  pure  cultures  mixed  with  the  food,  and  are  not  injured 
by  the  injection  of  blood  from  typhoid  patients.  Gaffky  failed 
completely  to  produce  any  symptoms  suggestive  of  typhoid  fever 
in  rabbits,  guinea-pigs,  white  rats,  mice,  pigeons,  chickens,  and 
calves,  and  found  that  Java  apes  could  feed  daily  upon  food  pol- 
luted with  typhoid  bacilli  for  a  considerable  time,  yet  without 
symptoms.  Griinbaum**  produced  typhoid  fever  in  chimpanzees 
by  inoculating  them  with  the  bacillus.  The  introduction  of  viru- 
lent cultures  into  the  abdominal  cavity  of  animals  is  followed  by 
peritonitis. 

Germano  and  Maureaff  found  that  mice  succumbed  in  from  one  to 
three  days  after  intraperitoneal  injection  of  i  or  2  cc.  of  a  twenty- 
four-hour-old  bouillon  culture.  Subcutaneous  injections  in  rabbits 
and  dogs  caused  abscesses. 

Losener  found  the  introduction  of  3  mg.  of  an  agar-agar  culture 
into  the  abdominal  cavity  of  guinea-pigs  to  be  fatal. 

PetruschkyJt  found  that  mice  convalescent  from  subcutaneous 
injections  of  typhoid  cultures  frequently  suffered  from  a  more  or  less 
widespread  necrosis  of  the  skin  at  the  point  of  injection. 

Prophylaxis. — One  of  the  most  important  and  practical  points 
for  the  physician  to  grasp  in  relation  to  the  subject  of  typhoid  fever 
is  the  highly  infective  character  of  the  discharges,  bothfeces  and  urine. 

*  "Archiv.  de  physiol.  norm.  et.  path.,"  1887. 

t  "Deutsche  med.  Wochenschrift,"  1899,  xv,  xvi. 

j"Sitz.  der  k.  k.  Gesellschaft  d.  Aerzt.  in  Wien,"  "Aerztl.  Central-Anz.," 
1898,  No.  3. 

§  "Jour.  Amer.  Med.  Assoc.,"  Aug.  28,  1897. 

||  "Bull.  Johns  Hopkins  Hospital,"  Dec.,  1897. 
"*  "Brit.  Med.  Jour.,"  April  9,  1904. 
ft  "Ziegler's  Beitrage,"  Bd.  xn,  Heft  3,  p.  494. 
ifZeitschrift  fur  Hygiene,"  1892,  Bd.  xn,  p.  261. 


600  Typhoid  Fever 

In  every  case  the  greatest  care  should  be  taken  for  their  proper 
disinfection,  a  rigid  attention  paid  to  all  the  details  of  cleanliness  in 
the  sick-room,  and  the  careful  sterilization  of  all  articles  which  are 
soiled  by  the  patient.  If  country  practitioners  were  as  careful  in  this 
particular  as  they  should  be,  the  disease  would  be  much  less  frequent 
in  regions  remote  from  the  filth  and  squalor  of  large  cities  with  their 
unmanageable  slums,  and  the  distribution  of  the  bacilli  to  villages 
and  towns,  by  milk,  and  by  watercourses  polluted  in  their  infancy, 
might  be  checked. 

In  large  cities  where  typhoid  fever  has  been  endemic  the  incidence 
of  the  disease  has  been  enormously  reduced  by  purification  of  the 
water-supply.  Where  this  measure  is  not  possible,  the  safety  of  the 
individual  citizens  can  be  promoted  by  using  bottled  pure  waters 
for  drinking  purposes  or  by  boiling  the  water  for  domestic  con- 
sumption. 

In  military  camps,  etc.,  the  fly  as  a  carrier  of  the  infection  must 
first  be  excluded  from  the  latrines  and  then  as  well  from  the  kitchens 
and  mess  tents.  When  epidemics  are  in  progress,  green  vegetables 
and  oysters  that  may  be  polluted  by  infected  water  must  be  guarded 
against. 

Prophylactic  Vaccination. — Following  the  principle  of  Haffkine's 
anticholera  inoculations,  Pfeiffer  and  Kolle,*  Wright,  f  and  Wright 
and  Semplef  have  used  subcutaneous  injections  of  sterilized  cultures 
as  a  prophylactic  measure.  One  cubic  centimeter  of  a  bouillon  culture 
sterilized  by  heat  was  used. 

The  "Indian  Medical  Gazette"  gives  the  following  important 
figures  showing  what  was  accomplished  in  1899:  Among  the  British 
troops  in  India  there  were  1312  cases  of  typhoid  fever,  with  348 
deaths  (25  per  cent.).  The  ratio  of  admissions  to  the  total  strength 
was  20.6  per  1000.  There  were  4502  inoculations,  and  among  them 
there  were  only  9  deaths  fromv typhoid  fever — 0.2  per  cent,  of  the 
strength.  There  were  44  admissions,  giving  0.98  per  cent,  of  the 
strength.  Among  the  non-inoculated  men  of  the  same  corps  and  at 
the  same  stations,  of  25,851  men  there  were  675  cases  and  146 
deaths,  giving  the  relative  percentages  of  admissions  and  deaths  as 
2.54  and  o.56.§ 

In  a  later  contribution,  Wright ||  showed  that  the  prophylactic 
vaccination  against  typhoid  fever  reduced  the  number  of  cases  and 
diminished  the  death-rate  among  the  inoculated,  and  also  called 
attention  to  the  slight  risk  the  inoculated  run  of  being  injured  in 
case  their  vital  resistance  is  below  normal,  or  they  are  already  in  the 
early  stages  of  the  disease,  or  where  the  dose  administered  is  too 
large,  or  the  second  vaccination  given  too  soon  after  the  first. 

*  "Deutsche  med.  Wochenschrift,"  1896,  xxn;  1898,  xxiv. 
'  "Lancet,"  Sept.,  1896. 

"Brit.  Med.  Jour.,"  1897,  i,  p.  256. 

"Phila.  Med.  Jour.,"  Oct.  13,  1900,  p.  688. 

"The  Lancet,"  Sept.  6,  1902. 


Specific  Therapy  601 

In  1903  Wright*  published  new  statistics  on  the  subject,  and 
between  1903  and  1908  numerous  references  to  the  subject  appear 
in  the  "British  Medical  Journal,"  in  the  "Lancet,"  and  in  the 
"Journal  of  the  Royal  Army  Medical  Corps,"  all  favorable  in  their 
general  attitude. 

During  the  Mexican  Revolution  of  1911,  the  United  States  Govern- 
ment began,  on  March  10,  1911,  the  mobilization  of  regiments  of  the 
United  States  Army  on  the  Mexican  frontier  near  San  Antonio, 
Texas.  In  order  to  prevent  repetition  of  the  sad  experiences  of  the 
Spanish-American  War,  in  which  the  troops  suffered  terribly  from 
typhoid  fever,  the  Secretary  of  War  determined  that  the  entire 
command  should  be  immunized  against  the  disease.  Many  of  the 
soldiers  arriving  on  the  ground  had  already  been  immunized,  the  re- 
mainder were  at  once  given  the  necessary  injection  upon  arrival. 
The  mean  strength  of  the  command  at  San  Antonio  was  12,000  up 
to  June  30,  1911,  a  period  approximating  four  months.  During  all 
that  time  there  were  only  2  cases  of  typhoid  fever  in  the  encamp- 
ment, i  in  an  uninoculated  civilian  teamster  and  i  in  an  inoculated 
soldier.  Both  cases  recovered.  The  soldier  suffered  from  so  mild 
an  attack  that  it  would  not  have  been  diagnosed  had  not  a  blood- 
culture  been  made.  During  the  four  months  from  March  loth 
to  June  3oth  the  typhoid  fever  was  prevalent  among  the  civilians 
of  San  Antonio,  there  being  40  cases  with  19  deaths,  f 

The  prophylactic  used  was  prepared  from  a  specially  selected 
strain  of  Bacillus  typhosus  grown  on  agar-agar  in  Kolle  flasks  for 
twenty-four  hours.  The  growth  was  washed  off  with  normal  salt 
solution,  killed  by  heating  at  55°  to  56°C.  in  a  water-bath,  standard- 
ized by  counting  the  bacteria  according  to  the  method  of  Wright, 
and  after  being  diluted  with  salt  solution,  0.25  per  cent,  of  trikresol 
was  added.  One  cubic  centimeter  of  the  finished  prophylactic  con- 
tained 1,000,000,000  bacilli.  The  first  dose  injected  contained 
500,000,000  bacilli,  the  second  and  third,  given  after  ten  and  twenty 
days,  contained  1,000,000,000  each.  The  injections  caused  little 
inconvenience  either  locally  or  constitutionally.  Only  i  case  had 
fever,  chills,  and  sweats,  and  this  was  the  only  case  requiring 
treatment  in  the  hospital.  It  subsequently  developed  that  this 
soldier  was  suffering  from  early  tuberculosis,  which  may  explain  the 
untoward  symptoms  from  which  he  suffered. 

Specific  Therapy. — Animals  can  be  immunized  to  this  bacillus,  and 
then,  according  to  Chantemesse  and  Widal,  develop  antitoxic  blood 
capable  of  protecting  other  animals.  Sternf  found  in  the  blood  of 
human  convalescents  a  substance  thought  to  have  a  protective  effect 
upon  infected  guinea-pigs.  His  observation  is  in  accordance  with  a 

*  "Brit.  Med.  Jour.,"  Oct.  10,  1903. 

t  "  Report  of  the  Surgeon-General  of  the  United  States  Army  to  the  Secretary 
of  War,"  IQII,  Washington,  D.  C. 

J  "Zeitschrift  fur  Hygiene,"  1894,  xvi,  p.  458. 


602 


Typhoid  Fever 


previous  one  by  Chantemesse  and  Widal,  and  has  recently  been 
abundantly  confirmed. 

The  immunization  of  dogs  and  goats  by  the  introduction  of 
increasing  doses  of  virulent  cultures  has  been  achieved  by  Pfeiffer 
and  Kolle*  and  by  Lofifler  and  Abel.f  From  these  animals  immune 
serums  were  secured. 

WalgerJ  reported  4  cases  treated  successfully  with  a  serum  ob- 
tained from  convalescent  patients.  Ten  cubic  centimeters  were 
given  at  a  dose,  and  the  injection  was  repeated  in  i  case  with  relapse. 

Rumpf§  and  Kraus  and  Buswell||  report  a  number  of  cases  of 
typhoid  favorably  influenced  by  hypodermic  injections  of  small  doses 
of  sterilized  cultures  of  Bacillus  pyocyaneus. 

Jez**  believes  that  the  antitoxic  principle  in  typhoid  fever  is  con- 
tained in  some  of  the  internal  organs  instead  of  the  blood,  and  claims 


Fig.  251. — Typhoid  bacilli,  unaggluti- 
nated  (Jordan). 


Fig.  252. — Typhoid  bacilli,  showing 
typical  clumping  by  typhoid  serum 
(Jordan). 


to  have  obtained  remarkable  results  in  18  cases  treated  with  extracts 
of  the  bone-marrow,  spleen,  and  thymus  of  rabbits  previously  in- 
jected with  the  typhoid  bacillus. 

Chantemesse,  ff  Pope,JJ  and  Steele§§  have  all  used  serums  from 
animals  immunized  against  typhoid  cultures  for  the  treatment  of 
typhoid  fever,  with  more  or  less  success  but  an  analysis  of  the  results 
shows  them  to  be  very  inconclusive. 

The   serum  prepared    by    Macfadyen,H||    by    crushing    cultures 

"Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Jan.  23,  1896,  Bd.  xix,  No.  23,  p.  51. 
Ibid,  1896. 

'Miinchener  med.  Wochenschrift,"  Sept.  27,  1898. 

'Deutsche  med.  Wochenschrift,"  1893,  No.  41. 

'Wiener  klin.  Wochenschrift,"  July  12,  1894. 

'Med.  moderne,"  March  25,  1899. 

'Gaz.  des  Hopitaux,"  1898,  LXXI,  p.  397. 

'Brit.  Med.  Jour.,"  1897,  i,  259. 

Ibid.,  April  17,  1897. 

"Brit.  Med.  Jour.,"  April  3,  1903. 


Bacteriologic  Methods  603 

frozen  with  liquid  air  and  injecting  animals  with  the  thus  liberated 
intracellular  toxin,  seems  to  be  no  improvement  upon  others. 

Meyer  and  Bergell*  prepared  a  serum  by  injecting  horses  with  a 
new  typhoid  toxin.  After  two  years'  treatment  they  were  able  to 
demonstrate  its  value  in  curing  infection  in  laboratory  animals, 
von  Leydenf  speaks  in  favorable  terms  of  this  serum. 

The  typhoid  immune  (bacteriolytic)  serum  is  specific,  but  its 
action  requires  the  presence  of  additional  complementary  substance, 
and  by  itself  it  is  useless.  Indeed,  it  may  do  harm  by  causing  the 
formation  of  anti-immune  bodies. 

So  far  no  serum  has  been  produced  that  is  of  any  certain  value  in 
therapeutics. 

Bacteriologic  Diagnosis. — There  are  four  bacteriologic  methods 
that  may  assist  the  clinician  in  completing  the  diagnosis  of  typhoid 
fever.  In  the  order  of  their  general  usefulness  and  practicability 
these  are: 

1.  The  Widal  reaction  of  agglutination. 

2.  The  blood-culture. 

3.  The  isolation  of  the  bacillus  from  the  feces. 

4.  The  conjunctival  and  dermal  reactions. 

Widal  Reaction  of  Agglutination. — This  very  valuable  adjunct  to 
our  means  of  making  the  diagnosis  of  atypical  and  obscure  cases  of 
typhoidal  infection  was  discovered  in  1896  when  Widal  and  Griin- 
baum,t  working  independently,  observed  that  when  blood-serum 
from  typhoid  fever  patients  is  added  to  cultures  of  the  typhoid 
bacillus  a  definite  reactive  phenomenon  occurs.  The  phenomenon, 
now  familiarly  known  as  the  "  Widal  reaction,"  consists  of  complete 
loss  of  the  motion  so  characteristic  of  the  typhoid  bacillus,  and  the 
collection  of  the  micro-organisms  into  clusters  or  groups — agglutina- 
tion. The  bacteria  frequently  appear  shrunken  and  partly  dissolved. 

The  technic  of  the  test  is  outlined  in  the  section  upon  Agglutination 
(q.v.).  For  the  use  of  the  practising  physician,  commercial  houses 
now  furnish  various  outfits  known  as  "  agglutometers,"  in  which  are 
found  such  simple  apparatus  and  directions  as  will  enable  those 
inexpert  in  laboratory  manipulations  to  arrive  at  fairly  accurate 
results. 

The  Blood-culture. — The  technic  of  this  operation  is  simple. 
The  skin  of  the  fold  of  the  elbow  is  thoroughly  cleansed,  a  fillet  put 
about  the  arm,  and  as  the  veins  become  prominent,  a  sterile  hypo- 
dermic needle  is  introduced  into  one  and  about  10  cc.  of  blood 
drawn  into  a  Keidel  tube  or  into  a  syringe.  Before  clotting  can  take 
•place,  this  is  discharged  into  a  small  flask  containing  100  cc.  of 
bouillon,  mixed,  and  stood  away  to  incubate.  After  twenty-four 
hours  the  bacilli  can  usually  be  found  in  pure  culture. 

*  "Med.  Klinik,"  in,  No.  31,  p.  917,  Aug.  4,  1907. 
t  "  Berl.  klin.  Wochenschrift,"  1907,  No.  18. 
t  "La  Semaine  Medicale,"  1896,  p.  295. 


604  Typhoid  Fever 

In  case  the  culture  is  not  pure,  the  typhoid  bacillus  can  be  sepa- 
rated from  contaminating  organisms  by  plating. 

The  Isolation  of  the  Bacillus  from  the  Feces. — This  method  of 
making  the  diagnosis  has  practically  been  abandoned  because  of  its 
uncertainty,  its  cumbersomeness,  its  tediousness,  and  because  the 
preceding  methods  suffice  in  all  cases. 

An  excellent  resume  of  the  many  methods  employed  for  isolating 
the  bacillus  from  the  stools  has  been  published  by  Peabody  and 
Pratt,*  and  is  appropriate  reading  for  those  interested  in  this 
subject. 

The  Conjunctival  Reaction. — An  additional  aid  to  the  diagnosis  of 
typhoid  in  doubtful  cases  based  upon  the  Wolff-Eisner-Calmette 
reaction  in  tuberculosis  is  the  "ocular  typhoid  reaction"  of  Chan- 
temesse.f  This  test  consists  in  the  instillation  into  the  eye  of  a 
solution  made  by  extracting  the  typhoid  bacillus  as  follows:  "  Gela- 
tin plates  covered  with  an  eighteen-  to  twenty-hour-old  culture  of 
virulent  typhoid  bacilli  were  washed  with  4  to  5  cc.  of  sterile  water. 
The  suspension  thus  obtained  was  heated  to  6o°C.,  centrifugated, 
and  the  supernatant  fluid  withdrawn.  The  centrifugated  organisms 
were  then  dried  and  triturated.  A  second  suspension  of  these 
broken  up  bacillary  bodies  was  then  made,  and  allowed  to  stand  for 
from  two  to  three  days  at  6o°C.  The  extract  thus  obtained,  after 
removing  the  disintegrated  and  digested  remnants,  was  precipitated 
with  alcohol,  forming  a  fine  coagulum.  This  was  subsequently 
dried,  powdered  and  dissolved  in  sterile  water  in  the  proportion 
of  0.02  mg.  to  a  drop."J 

When  one  drop  of  this  is  placed  upon  the  conjunctiva  of  a  patient 
in  the  early  days  of  typhoid  fever,  diffuse  redness  increases  and 
becomes  marked  in  two  or  three  hours.  '  There  is  also  some  feeling  of 
heat  in  the  eye.  Tears  flow  freely,  and  there  is  a  slight  mucopuru- 
lent  exudate  in  some  cases.  The  reaction  persists  about  ten  hours 
and  then  declines,  usually  disappearing  in  twenty-four  hours.  Ham- 
burger §  confirmed  the  results  of  Chantemesse.  It  is  too  early  to  say 
how  useful  the  reaction  is,  but  it  seems  to  promise  aid  in  diagnosing 
difficult  cases. 

Differential  Diagnosis  of  the  Typhoid  and  Colon  Bacilli.— This 
constitutes  the  chief  perplexity  of  bacteriologic  work  with  the  typhoid 
bacillus,  and  is  the  great  bugbear  of  beginners.  A  great  deal  of 
energy  has  been  expended  upon  it,  a  considerable  literature  has  been 
written  about  it,  and  much  still  remains  to  be  learned  by  which  it  may 
be  simplified. 

Two  chief  methods  are  in  vogue  at  present : 

1.  The  serum  differentiation. 

2.  The  culture  differentiation. 

"Boston  Medical  and  Surgical  Journal,"  1907. 

"Deutsche  med.  Wochenschrift,"  1907,  No.  31,  p.  1264. 
|  See  Hamburger,  "Jour.  Amer.  Med.  Assoc.,"  L,  17,  p.  1344,  April  25,  1008. 
§  Loc.  cit. 


Differentiation  of  Typhoid  and  Colon  Bacilli  605 

Serum  Differentiation. — The  specific  agglutinating  action  of 
experimentally  prepared  serums  can  be  used  to  differentiate  cultures 
of  the  colon,  paracolon,  typhoid,  and  paratyphoid  bacilli,  the  typhoid 
bacilli  alone  exhibiting  the  specific  effect  of  the  typhoid  serum.  This 
is  a  very  reliable  means  of  differentiation  when  the  cultures  have 
already  been  isolated.  The  method  is  described  under  the  heading 
"Agglutination,"  in  the  section  devoted  to  the  "  Special  Phenomenon 
of  Infection  and  Immunity." 

Richardson*  has  found  it  very  convenient  to  saturate  filter-paper  with  typhoid 
serum',  dry  it,  cut  into  0.5  cm.  squares,  and  keep  it  on  hand  in  the  laboratory  for 
the  purpose  of  making  this  differentiation.  To  make  a  test,  one  of  these  little 
squares  is  dropped  in  0.5  cc.  of  a  twenty-four-hour-old  bouillon  culture  of  the 
suspected  bacillus  and  allowed  to  stand  for  five  minutes.  A  drop  of  the  fluid 
placed  upon  a  slide  and  covered  will  then  show  typical  agglutinations  if  the 
culture  be  one  of  the  typhoid  fever  bacillus.  In  a  second  mention  of  this  method t 
he  has  found  its  use  satisfactory  in  practice  and  the  paper  serviceable  after  four- 
teen months'  keeping. 

The  Cultural  Differentiation. — When  the  typhoid  bacilli  are  to  be 
isolated  from  the  blood  of  living  patients,  they  are  so  likely  to  be 
obtained  in  pure  culture  that  little  trouble  is  experienced.  If  they 
are  to  be  isolated  from  the  pus  of  a  posttyphoidal  abscess,  or  from 
viscera  at  autopsy,  from  water  suspected  of  pollution,  and  especially 
when  they  are  to  be  isolated  from  the  intestinal  contents,  with  its 
rich  bacterial  flora,  the  matter  becomes  progressively  complicated. 

As  the  colonies  of  the  typhoid  bacilli  closely  resemble  those  of 
Bacillus  coli,  etc.,  special  media  have,  from  time  to  time,  been 
devised  for  the  purpose  of  emphasizing  such  differences  as  rapidity  of 
growth,  acid  production,  etc.  Thus,  Eisner  J  has  suggested  the 
employment  of  a  special  medium  made  as  follows: 

One  kilogram  of  grated  potatoes  (the  small  red  German  potatoes  are  best)  is 
permitted  to  macerate  over  night  in  i  liter  of  water.  The  juice  is  carefully 
pressed  out  and  filtered  cold,  to  get  rid  of  as  much  starch  as  possible.  The  filtrate 
is  boiled  and  again  filtered.  The  next  step  is  a  neutralization,  for  which  Eisner 
used  litmus  as  an  indicator,  and  added  2.5  to  3  cc.  of  a  ^f  o  normal  sodium 
hydrate  solution  to  each  10  cc.  of  the  juice.  Abbott  prefers  to  use  phenol- 
phthalein  as  an  indicator.  The  final  reaction  should  be  slightly  acid.  Ten  per 
cent,  of  gelatin  (no  peptone  or  sodium  chlorid)  is  dissolved  in  the  solution,  which 
is  boiled,  and  must  then  be  again  neutralized  to  the  same  point  as  before.  After 
filtration  the  medium  receives  the  addition  of  i  per  cent,  of  potassium  iodid;  then 
it  is  filled  into  tubes  and  sterilized  like  the  ordinary  culture-media. 

When  water  or  feces  suspected  to  contain  the  typhoid  bacillus  are  mixed  in 
this  medium  and  poured  upon  plates,  no  bacteria  develop  well  except  the  typhoid 
and  colon  bacilli. 

These,  however,  differ  markedly  in  appearance,  for  the  colon  colonies  appear 
of  the  usual  size  in  twenty-four  hours,  at  which  time  the  typhoid  bacillus,  if 
present,  will  have  produced  no  colonies  discoverable  by  the  microscope. 

It  is  only  after  forty-eight  hours — long  after  the  colon  colonies  have  become 
conspicuous — that  little  colonies  of  the  typhoid  bacillus  appear  as  finely  granular, 
small,  round,  shining,  dew-like  points,  in  marked  contrast  to  their  large,  coarsely 
granular  predecessors. 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1897,  p.  445. 

t  "Journal  of  Experimental  Medicine,"  May,  1898,  p.  353,  note. 

j  "Zeitschrift  fur  Hygiene,"  1895,  xxii,  Heft  i;  Dec.  6,  1896. 


606  Typhoid  Fever 

Unfortunately,  many  of  the  small  colonies  that  develop  in  Eisner's 
medium  subsequently  prove  to  be  those  of  the  colon  bacillus,  and 
the  method  is  thus  rendered  unreliable. 

Remy*  prefers  to  make  an  artificial  medium  approximating  a 
potato  in  composition,  but  without  dextrin  or  glucose.  The  com- 
position is  as  follows: 

Distilled  water 1000.0    grams 

Asparagin 6.0 

Oxalic  acid 0.5 

Lactic  acid 0.15 

Citric  acid °-I5 

Disodic  phosphate 5.0 

Magnesium  sulphate 2.5 

Potassium  sulphate i .  25 

Sodium  chlorid. 2.0 

All  the  salts  excepting  the  magnesium  sulphate  are  powdered  in  a  mortar  and 
introduced  into  a  flask  with  the  distilled  water.  Thirty  grams  of  Witte's  or 
Grubler's  peptone  are  then  added  and  the  mixture  heated  in  the  autoclave  under 
pressure  for  one-quarter  hour.  As  soon  as  removed,  the  contents  are  poured  into 
another  flask  into  which  120  to  150  grams  of  gelatin  had  previously  been  placed. 
The  flask  is  shaken  to  dissolve  the  gelatin,  and  the  contents  then  made  slightly 
alkaline  with  soda  solution.  The  mixture  is  again  heated  in  the  autoclave  at 
no°C.,  for  one-quarter  hour,  then  acidified  with  a  one-half  normal  solution  of 
sulphuric  acid,  so  that  10  cc.  have  an  acidity  neutralized  by  0.2  cc.  of  one-half 
normal  soda  solution.  This  acidity  is  equal  to  0.5  cc.  sulphuric  acid  per  liter. 
After  shaking,  place  the  flask  in  a  steam  sterilizer  for  ten  minutes,  then  filter. 
When  filtered,  verify  the  acidity  of  the  medium,  correcting  if  necessary. 
Finally,  add  the  magnesium  sulphate,  dissolve,  dispense  in  tubes,  and  sterilize  by 
the  intermittent  method. 

At  the  moment  of  using,  put  into  each  tube  i  cc.  of  a  35  per  cent,  solution  of 
lactose  and  o.i  cc.  of  a  2.5  per  cent,  solution  of  carbolic  acid. 

Upon  this  medium  the  colonies  of  the  typhoid  and  colon  bacilli 
show  marked  differences.  The  colon  colonies  are  yellowish  brown, 
the  typhoid  colonies  bluish  white  and  small.  Fine  bubbles  of  gas 
from  the  fermentation  of  the  lactose  often  occur  about  the  colon 
colonies. 

By  this  method  Remy  was  able  to  isolate  the  typhoid  bacillus  from 
the  stools  in  23  cases  which  he  studied.  He  believes  that  the  con- 
stant presence  of  the  typhoid  bacillus  in  the  stools  of  typhoid  fever, 
and  its  absence  from  them  under  all  other  conditions,  is  a  far 
more  important  and  valuable  method  of  diagnosis  than  even  the 
Widal  reaction. 

Wiirtzf  and  KashidaJ  make  the  differential  diagnosis  by  observ- 
ing the  acid  production  of  Bacillus  coli  in  a  medium  consisting  of 
bouillon  containing  1.5  per  cent,  of  agar,  2  per  cent,  of  milk-sugar, 
i  per  cent,  of  urea,  and  30  per  cent,  of  tincture  of  litmus.  This  is 
the  so-called  litmus-lactose-agar-agar.  The  culture-medium  should 
be  blue.  When  liquefied,  inoculated  with  the  colon  bacillus,  poured 
into  Petri  dishes,  and  stood  for  from  sixteen  to  eighteen  hours  in  the 
incubator,  the  blue  color  passes  off  and  the  culture-medium  becomes 

*  "Ann.  de  1'Inst.  Pasteur,"  Aug.,  1900. 

t  "  Archiv.  de  med.  Experimental, "  1892,  iv,  p.  85. 

f'Centralbl.  f.  Bkt.  u.  Parasitenk,  June  24,  1897,"  Bd.  xxi,  Nos.  20  and  21. 


Differentiation  of  Typhoid  and  Colon  Bacilli  607 

red.  If  a  glass  rod  dipped  in  hydrochloric  acid  be  held  over  the  dish, 
vapor  of  ammonium  chlorid  is  given  off.  The  typhoid  bacillus  pro- 
duces no  acid  in  this  medium,  and  there  is  consequently  no  change  in 
its  color.  Upon  plates  with  colonies  of  both  bacilli,  the  typhoid 
colonies  produce  no  change  of  color,  while  the  colon  colonies  at  once 
redden  the  surrounding  medium. 

Rothberger*  first  employed  neutral  red  for  the  differentiation  of  the 
typhoid  and  colon  bacilli.  When  grown  in  fluid  media  containing  it, 
the  colon  bacillus  produces  a  yellowish  fluorescence,  while  the 
typhoid  bacillus  does  not  destroy  the  port- wine  color.  Savage f  and 
Irons i  have  made  use  of  the  color  reaction  for  the  routine  detection  of 
the  colon  bacillus  in  water.  The  best  adaptation  of  the  method  is  by 
Stokes,  §  who  adds  it  to  the  various  sugar  bouillons  in  the  propor- 
tion of  o.i  gram  per  liter,  and  uses  the  medium  in  the  fermentation 
tube.  The  colon  bacillus  always  ferments  the  sugars  and  produces  a 
typical  color  reaction. 

Hiss ||  recommends  the  use  of  two  special  media. 

The  first  consists  of  5  grams  of  agar-agar,  80  grams  of  gelatin,  5  grams  of  Liebig's 
beef-extract,  5  grams  of  sodium  chlorid,  and  10  grams  of  glucose  to  the  liter. 
The  agar  is  dissolved  in  the  1000  cc.  of  water,  to  which  have  been  added  the  beef- 
extract  and  sodium  chlorid.  When  the  agar  is  completely  melted,  the  gelatin  is 
added  and  thoroughly  dissolved  by  a  few  minutes'  boiling.  The  medium  is  then 
titrated  to  determine  its  reaction,  phenolphthalein  being  used  as  the  indicator, 
and  enough  HC1  or  NaOH  added  to  bring  it  to  the  desired  reaction — i.e.,  a  reac- 
tion indicating  1.5  per  cent,  of  normal  acid.  To  the  clear  medium  add  one  or 
two  eggs,  well  beaten  in  25  cc.  of  water;  boil  for  forty-five  minutes,  and  filter 
through  a  thin  layer  of  absorbent  cotton.  Add  the  glucose  after  clearing. 

This  medium  is  used  in  tubes,  in  which  the  culture  is  planted  by  the  ordinary 
puncture.  The  typhoid  bacilhis  alone  has  the  power  of  uniformly  clouding  this 
medium  without  showing  streaks  or  gas-bubbles. 

The  second  medium  is  used  for  plating.  It  contains  10  grams  of  agar,  25 
grams  of  gelatin,  5  grams  of  beef-extract,  5  grams  of  sodium  chlorid,  and  10 
grams  of  glucose.  The  method  of  preparation  is  the  same  as  for  the  tube-me- 
dium, care  always  being  taken  to  add  the  gelatin  after  the  agar  is  thoroughly 
melted,  so  as  not  to  alter  this  ingredient  by  prolonged  exposure  to  high  tempera- 
ture. The  preparation  should  never  contain  less  than  2  per  cent,  of  normal  acid. 
Of  all  the  organisms  upon  which  Hiss  experimented  with  this  medium,  Bacilhis 
typhosus  alone  displayed  the  power  of  producing  thread- forming  colonies. 

The  colonies  of  the  typhoid  bacillus  when  deep  in  Hiss'  medium  appear  small, 
generally  spheric,  with  a  rough,  irregular  outline,  and,  by  transmitted  light,  of  a 
vitreous  greenish  or  yellowish-green  color.  The  most  characteristic  feature  con- 
sists of  well-defined  filamentous  outgrowths,  ranging  from  a  single  thread  to  a 
complete  fringe  about  the  colony.  The  young  colonies  are,  at  times,  composed 
solely  of  threads'.  The  fringing  threads  generally  grow  out  nearly  at  right  angles 
to  the  periphery  of  the  colony. 

The  colonies  of  the  colon  bacillus  appear,  on  the  average,  larger  than  those  of 
the  typhoid  bacillus;  they  are  spheric  or  of  a  whetstone  form,  and  by  transmitted 
light  are  darker,  more  opaque,  and  less  refractive  than  the  typhoid  colonies.  By 
reflected  light  they  are  pale  yellow  to  the  unaided  eye. 

Surface  colonies  are  large,  round,  irregularly  spreading,  and  are  brown  or  yel- 
lowish-brown in  color.  Hiss  claims  that  by  the  use  of  these  media  the  typhoid 
bacillus  can  readily  be  detected  in  typhoid  stools. 

*  "Centralbl.  f.  Bakt,"  1893,  P-  187. 

t  "Journal  of  Hygiene,"  1901,  i,  p.  437. 

j  Ibid.,  1902,  n,  p.  437. 

§  "Jour,  of  Infectious  Diseases,"  1904,  i,  p.  341. 

||  "Jour,  of  Experimental  Medicine,"  Nov.,  1897,  vol.  n,  No.  6. 


608  Typhoid  Fever 

Piorkowski*  recommends  a  culture-medium  composed  of  urine  two 
days  old,  to  which  0.5  per  cent,  of  peptone  and  3.3  per  cent,  of 
gelatin  have  been  added.  Colonies  of  the  typhoid  bacillus  appear 
radiated  and  filamentous;  those  of  the  colon  bacillus,  round,  yellow- 
ish, and  sharply  defined  at  the  edges.  The  cultures  should  be  kept  at 
22°C.,  and  the  colonies  should  appear  in  twenty-four  hours. 

Adami  and  Chapin*  have  suggested  a  method  for  the  isolation  of 
typhoid  bacilli  from  water,  in  which  use  is  made  of  the  agglutination 
of  the  bacilli  by  immune  serum. 

Two  quart  bottles  (Winchester  quarts)  are  carefully  sterilized  and  filled  with 
the  suspected  water  with  an  addition  of  25  cc.  of  nutrient  broth  and  incubated 
for  eighteen  to  twenty-four  hours  at  37°C.  By  this  time  the  typhoid  bacillus 
grows  abundantly  in  spite  of  the  small  amount  of  nourishment  the  water  contains. 
At  the  end  of  the  incubation,  10  cc.  of  the  fluid  is  filled  into  each  of  a  number  of 
long  narrow  (7  mm.)  test-tubes  made  by  sealing  a  glass  tube  one-half  meter  long 
at  one  end.  About  i  inch  from  the  bottom  the  tube  is  filed  completely  round 
so  as  to  break  easily  at  that  point.  The  different  tubes  next  receive  additions  of 
typhoid  immune  serum  sufficient  to  make  the  dilutions  1:60,  1:100,  1:150,  and 
i :  200.  If  typhoid  bacilli  are  present,  within  a  quarter  of  an  hour  beginning 
agglutination  can  be  seen,  and  by  the  end  of  two  to  five  hours  flocculent  masses 
collect  at  the  bottom  of  the  tube,  forming  a  flocculent  precipitate.  The  next 
procedure  should  be  with  the  tube  showing  agglutination  with  the  greatest  dilu- 
tion, as  the  more  concentrated  preparations  carry  down  not  only  the  typhoid 
bacilli,  but  also  closely  related  organisms.  After  the  sedimentation  of  the  agglu- 
tinated bacilli  is  complete,  the  tube  is  broken  at  the  file  mark,  and  the  sediment 
contained  in  the  short  tube  washed  with  two  or  three  changes  of  distilled  water, 
being  allowed  to  settle  each  time.  This  removes  many  of  the  organisms  not 
agglutinated.  A  loopful  of  the  washed  sediment  is  transferred  to  a  tube  of 
nutrient  broth,  and  finally  from  this  tube  plate  cultures  are  made  upon  Eisner's  or 
Hiss'  media. 

A  culture-medium  for  isolating  the  typhoid  bacillus  from  feces  is 
recommended  by  Drigalski-Conradif  and  by  Petkowitsch.f  It  is 
made  as  follows: 

Horse-meat  infusion  (3  poimds  of  horse  meat  to  2 

liters  of  water) 2  liters 

Witte's  peptone 20  grams 

Nutrose 20  grams 

Sodium  chlorid 10  grams 

Agar-agar 60  grams 

Litmus  solution  (Kubel  and  Tiemann) 260  cc. 

Lactose 30  grams 

Crystal- violet  solution  (o.oi  per  cent.) 20  cc. 

Before  adding  the  crystal-violet  solution  render  feebly  alkaline  to  litmus 
(about  0.04  per  cent,  of  pure  soda). 

Colon  colonies  upon  this  medium  appear  in  fourteen  to  sixteen  hours  to  be  red 
and  opaque.  Typhoid  colonies  blue  or  violet,  transparent  and  drop-like. 

Beckman§  modifies  the  preparation,  making  it  as  follows: 

*  "Berliner  klin.  Wochenschrift,"  Feb.  13,  1899. 
t  "_Zeitschrift  f.  Hygiene,"  Bd.  xxrx. 

j  "Centralbl.  f.  Bakt.,"  etc.,  May  28,  1904,  Bd.  xxxvi,  No.  2,  p.  304. 
§  See  F.  F.  Wesbrook,  "Jour.  Infectious  Diseases,"  May,  1905,  Supplement, 
No.  i,  p.  319. 


Differentiation  of  Typhoid  and  Colon  Bacilli  609 


(a)  Add  i  liter  of  water  to  680  grams  of  finely  chopped  lean  beef  and  place  in 
the  cold  for  twenty-four  hours.  Express  the  juice  and  make  up  to  i  liter.  Coagu- 
late the  albumin,  either  by  boiling  for  ten  minutes  or  by  heating  to  i2o°C.  in 
the  autoclave.  Filter.  Add  10  grams  of  Witte's  peptone,  10  grams  of  nutrose, 
and  5  grams  of  sodium  chlorid.  Heat  in  the  autoclave  at  a  temperature  of 
i2o°C.  for  thirty  minutes,  or'boil  vigorously  for  fifteen  minutes.  Render  slightly 
alkaline  to  litmus  paper.  Filter.  Add  30  grams  of  agar.  Heat  in  the  autoclave 
at  a  temperature  of  i2o°C.  for  one-half  hour,  or  heat  over  the  gas-flame  until  the 
agar  is  dissolved.  Render  slightly  alkaline  to  litmus  paper  while  hot,  if  necessary. 
Filter  through  glass  wool  into  a  sterile  vessel. 

(6)  To  130  cc.  of  litmus  solution  (Kubel  and  Tiemann's)  add  15  grams  of 
chemically  pure  lactose.  Boil  for  ten  minutes. 

(c)  Mix  (a)  and  (b)  while  hot.  Render  slightly  alkaline  to  litmus,  if  necessary. 
To  the  mixture  add  2  cc.  of  hot  sterile  solution  of  10  per  cent,  sodium  hydrate 
in  distilled  water  and  10  cc.  of  a  fresh  solution  of  Hochst's  crystal  violet  (o.i 
gram  of  crystal  violet  to  100  cc.  of  sterile  water). 

The  medium  is  now   poured  into  Petri 

dishes  and  is  of  a  deep  purple  color.     So    r  i 

much  water  of  condensation  forms  on  the 
solidified  surface  that  it  is  an  advantage  to  use 
porous  clay  covers  (Hill)  for  the  Petri  dishes 
instead  of  the  ordinary  glass  covers.  The 
medium  keeps  well  but  dries  up  rapidly. 

A  very  ingenious  method  of  isolat- 
ing the  typhoid  and  colon  bacilli  from 
drinking  water  has  been  suggested  by 
Starkey,*  who  uses  a  tubular  laby- 
rinth of  glass  filled  with  ordinary  bouil- 
lon containing  0.05  per  cent,  of  car- 
bolic acid,  or,  as  recommended  by 
Somers,t  Pariette's  bouillon.  The 
original  formula  for  the  latter  medium 
is  as  follows: 

1.  Measure  out  pure  hydrochloric  acid, 

4  cc.,  and  add  it  to  carbolic  acid 

solution   (5  per  cent.),   100  cc.    Fig.  253.— Starkey 's  labyrinth  as 

Allow  the  solution  to  stand  at  least  modified  by  Somers. 

a  few  days  before  use. 

2.  This  solution  is  added  in  quantities  of  o.i,  0.2,  and  0.3  cc.  (delivered  by 

means  of  a  sterile  graduated  pipette  to  tubes,  each  containing  10  cc. 
of  previously  sterilized  nutrient  bouillon). 

3.  Incubate  at  37°C.  for  forty-eight  hours  to  eliminate  contaminated  tubes. 

The  restraining  medium  prevents  the  ready  growth  of  most  organisms 
except  colon  and  typhoid  bacilli.  The  anaerobic  conditions  prevent 
the  development  of  aerobic  organisms  which  form  the  majority  of 
bacteria  with  which  one  comes  in  contact  in  ordinary  bacteriological 
examinations.  The  typhoid  bacillus,  being  more  motile  than  the 
colon,  travels  more  quickly  through  the  coils  of  the  labyrinth  and  first 
arrives  at  its  end,  where  it  can  be  found  in  pure  or  nearly  pure  cul- 
ture after  about  forty-eight  hours. 

Somers  has  improved  the  labyrinth  by  bending  it  in  a  circular 

!  "  Amer.  Jour.  Med.  Sci.,"  July,  1906,  cxxxii,  No.  i,  No.  412,  p.  109. 
t  "Trans.  Phila.  Path.  Soc./'  1906. 
39 


610  Typhoid  Fever 

form,  so  that  it  can  stand  alone,  and  by  adapting  its  size  to  the  Novy 
jar,  so  that  satisfactory  anaerobic  conditions  can  easily  be  attained. 
Hesse*  has  recommended  the  following  medium: 

Agar-agar 5  grams  (4.5  grams  absolutely  dry). 

Witte's  peptone 10 

Liebig's  beef -extract 5 

Sodium  chlorid 8.5 

Distilled  water 1000 

Dissolve  the  agar-agar  in  500  cc.  of  the  water  over  a  free  flame,  making  up  the 
loss  by  evaporation.  Dissolve  the  other  ingredient,  in  the  remaining  500  cc. 
of  water,  heat  until  dissolved,  replacing  the  loss  by  evaporation.  Pour  the  two 
solutions  together,  heat  for  thirty  minutes  and  add  distilled  water  to  replace 
loss  by  evaporation.  Filter  through  cotton  until  clear.  Adjust  reaction  to  i 
per  cent,  acidity.  Tube — 10  cc.  to  a  tube.  Sterilize  in  the  autoclave. 

The  medium  is  used  for  plating.  The  material  containing  the 
micro-organisms  must  be  so  dilute  that  only  a  few  colonies  will 
develop  upon  the  plates.  The  typhoid  colonies  greatly  outgrow  the 
colon  colonies  and  may  attain  to  a  diameter  of  several  centimeters. 
They  show  a  small  opaque  center  and  an  opalescent  body  and  appear 
circular. 

Capaldif  recommends  the  following  medium  for  plating  typhoid 
and  colon  colonies: 

Witte's  peptone 20  grams 

Gelatin 10  " 

Agar-agar 20 

Dextrose  or  mannite 10  " 

Sodium  chlorid 5  " 

Potassium  chlorid 5  " 

Distilled  water 1000  " 

Dissolve  the  agar  in  500  cc.  of  water,  the  other  ingredients  in  the  other  500  cc. 
of  water.  Pour  together,  add  10  cc.  of  NaOH,  filter,  and  tube. 

Upon  this  medium  the  typhoid  colonies  are  small,  glistening, 
bluish,  and  translucent.  Colon  colonies  are  larger,  opaque,  and 
brownish. 

Endot  recommends  the  employment  of  the  following  medium  upon 
which  colonies  of  the  typhoid  bacillus  grow  large  and  remain  colorless 
while  those  of  the  colon  bacillus  remain  small  and  red: 

1000  cc.  of  meat  infusion. 
30  grams  of  agar-agar. 
10  grams  of  peptone  (Witte's). 
5  grams  of  sodium  chlorid. 

Neutralize  and  clear  by  nitration,  then  add  10  cc.  of  a  10  per  cent,  solution  of 
NaOH  to  alkalinize,  10  grams  of  chemically  pure  lactose  and  5  cc.  of  a  filtered, 
saturated,  alcoholic  solution  of  fuchsin.  Next  add  25  cc.  of  a  10  per  cent,  sodium 
sulphite_solution,  by  which  the  intense  red  given  by  the  fuchsin  is  entirely  bleached 
by  the  time  the  agar-agar  is  cold.  After  adding  the  necessary  reagents  and  while 
still  warm  and  perhaps  red,  tube  the  medium.  The  tubes  should  be  kept  in  the 
dark. 

*  "Zeitschrift  fur  Hygiene,"  1908,  LVIII,  441. 
"Zeitschrift  fur  Hygiene,"  1896,  xxm,  475. 
t  "Centralbl.  f.  Bakt.,"  etc.,  1904,  xxxv. 


Differentiation  of  Typhoid  and  Colon  Bacilli  611 

Loffler*  has  found  malachite  green  a  very  useful  adjunct  to  our 
means  of  differentiating  the  typhoid  from  other  similar  bacilli. 

For  the  purpose,  2  ^  to  3  per  cent,  of  a  2  per  cent,  solution  of  malachite  green 
are  added  to  the  culture-medium.  The  preparation  given  the  preference  con- 
sists of  i  pound  of  meat  macerated  in  i  liter  of  water,  neutralized  with  potassium, 
with  the  addition  of  2  per  cent,  of  peptone,  5  per  cent,  of  lactose,  i  per  cent,  of 
glucose,  0.5  per  cent,  of  sodium  sulphate,  2  per  cent,  of  nitrate  of  potassium,  and 
3  per  cent,  of  a  2  per  cent,  solution  of  malachite  green. 

In  the  medium  the  ordinary  cocci  and  bacilli  do  not  grow,  Gart- 
ner's bacillus  and  the  paratyphoid  bacillus  b  leave  the  medium  clear, 
but  grow  as  a  deposit  at  the  bottom  of  the  tube;  the  typhoid  bacillus 
destroys  the  green.  If  agar-agar  be  added,  the  colonies  are  sur- 
rounded by  a  clear  yellow  zone.  The  colon  and  other  organisms 
grow  slowly  if  at  all. 

Not  many  workers  were  satisfied  with  the  results  obtained  by 
malachite  green,  nor  were  the  results  obtained  uniform.  A  careful 
study  of  the  subject  was  made  by  Peabody  and  Pratt,  f  who  found 
great  differences  in  the  quality  and  reactions  of  different  malachite 
greens  in  the  market.  That  with  which  Loffler  worked  was  com- 
mercially known  as  "120."  They  obtained  three  samples  of  this 
dye,  which  varied  in  acidity  between  wide  margins  (0.2-1.0). 
Experimenting  with  the  different  preparations,  they  found  that  the 
least  acid  was  the  most  useful  preparation.  The  success  of  the 
method,  therefore,  depends  upon  the  adjustment  of  the  concentra- 
tion of  the  dye  to  the  reaction  of  the  medium.  When  this  is  done, 
malachite  green  becomes  a  valuable  adjunct  to  specific  differentia- 
tion. Their  studies  of  the  media  led  Peabody  and  Pratt  to  the  inven- 
tion of  a  new  method  of  isolating  typhoid  bacilli  from  the  feces. 
Instead  of  employing  malachite  green  agar-agar  directly  for  this 
purpose,  they  first  employ  malachite  green  bouillon  as  an  "enrich- 
ing" culture,  and  after  eighteen  to  twenty-four  hours'  growth  in  the 
incubator  inoculate  one  or  two  large  (20  cm.  diameter)  Drigalski- 
Conradi  plates,  from  which  the  colonies  can  subsequently  be  picked 
out. 

Bile  salts  were  first  employed  in  culture-media  by  LimbourgJ  and 
have  been  more  or  less  popular  ever  since,  though  for  differentiation 
of  typhoid  and  colon  bacilli  they  cause  occasional  disappointment. 

Buxton  and  Coleman§  prepare  a  medium  composed  of: 

Ox-bile 900  cc. 

Glycerin 100  cc. 

Peptone 20  grams 

This  was  placed  in  a  number  of  100  cc.  flasks,  sterilized  in  the  Arnold 
sterilizer,  and  employed  chiefly  for  blood-culture.  The  typhoid 
bacillus  grows  well  in  it. 

Boston  Med.  and  Surg.  Journal,"  Feb.  13,  1908,  CLVIII,  p.  213. 


t  "  Zeitschrif t  f.  physiol.  Chemie,"  1889,  m,  p.  196. 
t  "Inst.  hyg.  Univers.  Griefswald,"  see  "Bull.  Ii 


[nst.  Past.,"  rv,  No.  9,  May  15, 
1906,  p.  393. 

§  "Journal  of  Infectious  Diseases,"  1909,  vi,  No.  2,  p.  194. 


612  Typhoid  Fever 

Jackson*  prepares  a  medium  for  water  examination  when  typhoid 
and  colon  bacilli  are  suspected.  It  consists  of  undiluted  ox-bile  to 
which  i  per  cent,  of  peptone  and  i  per  cent,  of  lactose  are  added. 
It  is  filled  into  fermentation- tubes  of  40.  cc.  capacity  and  sterilized  in 
the  Arnold  apparatus.  If  fresh  ox-bile  cannot  be  secured,  an  1 1  per 
cent,  solution  of  dry  ox-bile  can  be  made;  10  cc.  of  suspected  water 
or  milk  are  planted  in  the  tubes  of  this  medium.  The  contained 
micro-organisms  grow  rapidly,  typhoid  bacilli  outgrowing  all 
others,  and  not  fermenting  the  sugar;  rapid  fermentation  and 
copious  gas-formation  take  place  if  colon  bacilli  are  present. 

An  excellent  medium  suggested  by  MacConkeyf  has  the  following 
composition : 

Agar 1.5  grams 

Sodium  taurocholate 0.5  gram 

Peptone! 2.0  grams 

Water 100 .  o  cc. 

It  is  boiled,  clarified,  and  filtered  as  usual,  then  receives  an  addition  of  i.o 
gram  of  lactose,  is  tubed,  and  then  sterilized  three  times  on  successive  days. 

For  determining  fermentation  by  colon  bacilli  the  same  investiga- 
tor advises  a  broth  composed  of : 

Sodium  taurocholate  (pure) 0.5  gram 

Peptone 2.0  grams 

Glucose 0.5  gram 

Water 100.0  cc. 

Boil,  filter,  add  sufficient  neutral  litmus,  fill  into  fermentation-tubes,  and  steril- 
ize at  ioo°C.  Colon  colonies  appear  red;  typhoid,  blue. 

In  a  careful  study  of  the  bile-salt  media  MacConkeyf  points  out  an 
error,  first  discovered  by  Theobald  Smith,  that  depends  upon  the 
alkali  production  of  the  colon  bacillus  in  the  absence  of  sugar.  If 
too  little  sugar  be  added  to  the  medium,  the  alkali  production  masks 
the  acid  production  unless  the  oxygen  be  removed,  and  red  colonies 
of  the  colon  bacillus  grown  upon  the  medium  may  in  time  turn  dis- 
tinctly blue.  It  becomes  obvious,  therefore,  that  the  medium 
should  be  as  neutral  as  possible  to  the  indicator  used.  After  trial  he 
found  neutral  red  preferable  to  litmus,  and  makes  the  medium  as 
follows: 

i.  A  stock  solution  is  made: 

Sodium    taurocholate    (commerciaj    from    ox-bile    and 

neutral  to  neutral  red) 0.5  per  cent. 

Peptone  (Witte's) 2 .  o  per  cent. 

Water  (distilled  or  tap) 100 .  o  cc. 

(As  calcium  0.03  per  cent,  is  favorable  to  the  growth  of  the  organisms, 
it  should  be  added  if  distilled  water  is  used.) 

The  ingredients  should  be  mixed,  steamed  in  a  steam  sterilizer  for  one  to  two 
hours,  filtered  while  hot,  allowed  to  stand  twenty-four  to  forty-eight  hours,  then 
filtered  cold  through  paper.  A  clear  solution  should  then  result,  which  will  keep 
indefinitely  under  proper  conditions.  The  various  bile-salt  media  are  prepared 

*  "Biological  Studies  of  the  Pupils  of  W.  T.  Sedgwick,"  1906,  University  of 
Chicago  Press. 

t  "The  Thompson-Yates  Laboratory  Reports,"  m,  p.  151. 
J  "Journal  of  Hygiene,"  190.8,  vm,  p.  322. 


Bacilli  Resembling  the  Typhoid  Bacillus  613 

from  this  stock  solution  by  adding  glucose,  0.5  per  cent.;  lactose,  i  per  cent.; 
cane-sugar,  i  per  cent.;  dulcit,  0.5  per  cent.;  adonit,  0.5  per  cent.,  or  inulin,  i 
per  cent.;  and  neutral  red  (i  per  cent,  solution),  0.25  per  cent.,  distributing  into 
fermentation-tubes  and  sterilizing  in  the  steamer  for  fifteen  minutes  on  each  of 
three  successive  days. 

Bile-salt  agar-agar  is  made  by  dissolving  2  per  cent,  of  agar-agar  in  the  stock 
fluid,  either  in  the  steamer  or  in  the  autoclave.  The  mixture  is  cleared  with  an 
egg,  filtered,  neutral  red  added  in  the  same  proportion  as  for  the  broth,  and  dis- 
tributed into  flasks  in  quantities  of  80  cc.  When  required  for  use,  the  fer- 
mentable substance  is  added  to  the  agar  in  the  flask,  and  the  whole  placed  in  a 
water-bath  or  steamer  (care  must  be  taken  not  to  heat  either  the  fluid  or  solid 
medium  beyond  ioo°C.).  When  melted,  the  agar  preparation  is  poured  into 
Petri  dishes,  allowed  to  solidify,  and  then  dried  in  an  incubator  or  warm  room, 
the  plate  being  placed  upside  down  with  the  bottom  detached  and  propped  up 
on  the  edge  of  the  cover.  It  is  necessary  that  the  surface  of  the  agar-agar  should 
not  be  too  wet,  lest  the  colonies  become  confluent,  nor  too  dry,  lest  the  growth 
be  stunted.  Inoculations  are  made  by  placing  a  loopful  of  the  material  to  be 
examined  on  the  center  of  one  plate,  and  rubbed  over  the  surface  with  a  bent 
glass  rod;  the  same  rod,  without  recharging,  being  used  to  inoculate  the  surface 
of  two  other  plates.  The  plates  are  then  incubated  upside  down.  The  colonies 
of  the  colon  bacillus  appear  yellow. 

BACILLI  RESEMBLING  THE  TYPHOID  BACILLUS 

Bacillus  typhosus  is  one  of  a  group  of  organisms  possessing  a  con- 
siderable number  of  common  characteristics,  each  member  of  which, 
however,  can  be  differentiated  by  some  one  fairly  well-marked  pecu- 
liarity. At  one  end  of  the  series  is  the  typhoid  bacillus,  which  we 
conceive  to  be  devoid  of  the  power  to  liquefy  gelatin,  ferment  sugars, 
form  indol,  coagulate  milk,  or  progressively  form  acids.  At  the  other 
extreme  stands  Bacillus  coli,  an  organism  whose  typical  representa- 
tives coagulate  milk,  form  indol,  ferment  dextrose,  lactose,  sacchar- 
ose, and  maltose  with  the  formation  of  hydrogen  and  carbon  dioxid 

in  the  proportion  of  =^~  =  — 
CO2       i 

Between  these  extremes  are  numerous  organisms  known  as  " inter- 
mediates." It  is  usually  a  simple  matter  to  differentiate  these  forms 
from  the  typical  species  at  the  two  ends  of  the  series,  but  it  is  quite 
difficult  to  differentiate  them  from  one  another.  Whether  they  are 
of  sufficient  importance  to  make  it  worth  while  to  pay  much  atten- 
tion to  them  is,  as  yet,  uncertain;  and,  indeed,  we  do  not  know 
whether  they  are  to  be  regarded  as  variations  from  the  type  species 
or  separate  and  distinct  organisms.  The  fact  that  some  of  them  are 
associated  with  serious  and  fatal  disorders — paracolon  bacillus  and 
bacillus  of  psittacosis — proves  them,  at  least,  to  be  important. 
Buxton*  summarizes  the  main  points  of  difference  as  follows: 

B.  coli  com- 
munis.  Intermediates.        B.  typhosus 

Coagulation  of  milk. -j- 

Production  of  indol -j- 

Fermentation    of    lactose    with 

gas + 

Fermentation    of    glucose    with 

gas +  + 

Agglutination  by  typhoid  serum.  —  —  + 

*  "Journal  of  Medical  Research,"  vol.  vm,  No.  i,  June,  1902,  p.  201. 


614  Bacilli  Resembling  the  Typhoid  Bacillus 

The  characteristics  of  the  three  groups  as  shown  by  the  fermenta- 
tion-test stand  thus:* 

Gas  upon  Gas  upon  Gas  upon 

dextrose.  lactose.  saccharose. 

Bacillus  typhosus — 

Intermediates + 

Bacillus  coli  communis + 

Bacillus  coli  communior +                         +  + 

Buxton  finds  those  pathogenic  for  man  clinically  divisible  into 
three  groups,  as  follows: 

(a)  The  Meat-poisoning  Group. — This  includes  Bacillus  enteritidis 
of  Gartner  and  others.     The  symptoms  begin  soon  after  eating  the 
poisonous  meat,  and  are  toxic.     Bacilli  quickly  invade  the  body. 
The  illness  continues  four  or  five  days,  after  which  recovery  is  quick. 
In  a  few  cases  death  has  occurred  on  the  second  or  third  day. 

(b)  The  Pneumonic  or  Psittacosis  Group. — Psittacosis  is  an  epi- 
demic infectious  disease  with  pneumonic  symptoms  and  a  high 
mortality.     Its  origin  has  been  traced  to  diseased  parrots,  and  from 
them  Nocard  isolated  Bacillus  psittacosis,  supposed  to  be  the  cause 
of  the  disease  in  man.     Later  epidemics  were  studied  by  Achard 
and  Bensaude. 

(c)  The  Typhoidal  Group. — The  organisms  to  be  included  in  this 
group  occasion  symptoms  closely  resembling  typhoid  fever,  though 
they  differ  biologically  from  the  typhoid  bacillus,  and  do  not  agglu- 
tinate with  typhoid  serums. 

It  is  thus  evident  that  some  of  the  intermediates  occasion  symp- 
toms resembling  typhoid  fever,  while  others  occasion  symptoms 
widely  differing  from  it.  It  is  suggested  that  to  the  former  the 
term  paratyphoid  bacilli  be  applied,  while  the  latter  are  known  as 
paracolon  bacilli. 

Although  Achard  and  Bensa-ude,f  and  Johnson,  Hewlett,  and 
LongcopeJ  have  studied  the  paratyphoid  infections,  Gwyn,§  Lib- 
man,||  and  others  the  paracolon  bacilli,  and  Gushing**  and  Durhamff 
have  made  comparative  studies  of  the  members  of  the  group,  it  is 
still  too  soon  to  regard  the  knowledge  attained  sufficient  to  warrant 
particular  mention  of  the  various  intermediate  and  related  organ- 
isms in  a  work  of  this  kind.  In  the  following  pages,  therefore, 
attention  will  be  devoted  only  to  the  more  important  organisms  of 
the  group. 

Hiss  and  Zinsser,  "Text-book  of  Bacteriology,"  1910,  p.  429. 
"Soc.  Med.,"  Nov.,  1896. 
Amer.  Jour.  Med.  Sci.,"  Aug.,  1902. 
"ohns  Hopkins  Bulletin,"  1898,  vol.  ix. 
ournal  of  Medical  Research,"  1902,  p.  168. 
ohns  Hopkins  Bulletin,"  1900,  vol.  xi. 


ft 


ournal  of  Experimental  Medicine,"  1901,  vol.  v,  p.  353. 


Bacilli  Resembling  the  Typhoid  Bacillus 


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6i6  Bacillus  Coli 


BACILLUS  COLI  COMMUNIS  (ESCHERICH) 

General  Characteristics. — A  motile,  flagellated,  non-sporogenous,  aerobic  and 
optionally  anaerobic,  non-chromogenic,  non-liquefying,  aerogenic,  saprophytic, 
occasionally  pathogenic  bacillus,  staining  by  the  ordinary  methods,  but  not  by 
Gram's  method.  It  produces  indol,  coagulates  milk,  and  produces  acids  and 
gases  from  dextrose,  lactose,  and  sucrose. 

This  micro-organism  was  first  isolated  from  human  feces  by 
Emmerich,*  in  1885,  who  thought  it  to  be  the  specific  cause  of 
Asiatic  cholera,  and  called  it  Bacillus  neapolitanus.  Many  have  since 
studied  it  until  it  has  now  become  one  of  the  best  known  bacteria. 

Distribution. — It  is  habitually  present  in  the  feces  of  animals,  and 
in  water  and  soil  contaminated  by  them.  Soon  after  birth  the 
organism  finds  its  way  into  the  alimentary  canal  and  permanently 


Fig.  254. — Bacillus  coli  (Migula). 

establishes  itself  in  the  intestine,  where  it  can  be  found  in  great 
numbers  throughout  the  entire  life  of  the  individual.  It  is  almost 
certainly  identical  with  Bacillus  pyogenes  fcetidus  of  Passet,  and  so 
closely  resembles  B.  acidi  lactici  that  Prescottf  believes  them  to  be 
identical.  It  may  also  be  identical  with  Bacillus  lactis  aerogenes, 
Bacillus  cavicida,  and  other  separately  described  species. 

Morphology. — The  bacillus  is  rather  variable,  both  size  and  form 
depending  to  a  certain  extent  upon  the  culture  medium  on  which  it 
grows.  It  measures  about  1-3  X  0.4-0.7  ju.  It  usually  occurs  in  the 
form  of  short  rods,  but  coccus-like  and  elongate  individuals  may 
be  found  in  the  same  culture.  The  bacilli  are  usually  separate 
from  one  another,  though  occasionally  joined  in  pairs,  are  actively 

*"  Deutsche  med.  Wochenschrift,"  1885,  No.  2. 

t  Society  of  American  Bacteriologists,  Dec.  31,  1902. 


Bacilli  Resembling  the  Typhoid  Bacillus  617 

motile,  and  provided  with  flagella,  which  are  variable  in  number, 
usually  from  four  to  a  dozen.  The  organisms  from  some  cultures 
swim  actively,  even  when  the  culture  is  some  days  old;  others  are 
sluggish  even  when  young  and  actively  growing,  and  still  other 
cultures  consist  of  bacilli  that  scarcely  move  at  all.  It  forms  no 
endospores. 

Staining. — The  bacillus  stains  well  with  the  aqueous  solutions  of 
the  anilin  dyes,  but  not  by  Gram's  method. 

Cultivation. — It  is  readily  cultivated  upon  the  ordinary  media. 

Colonies. — Upon  gelatin  plates  the  colonies  are  visible  in  twenty- 
four  hours.  Those  situated  below  the  surface  appear  round,  yellow- 
brown,  and  homogeneous.  As  they  increase  in  size  they  become 
opaque.  The  superficial  colonies  are  larger  and  spread  out  upon  the 


Fig.  255. — Bacillus  coli  communis;  superficial  colony  two  days  old  upon  a  gelatin 
plate.     X  21  (Heim). 

surface.  The  edges  are  dentate  and  slightly  resemble  grape-vine 
leaves,  often  showing  radiating  ridges  suggestive  of  the  veins  of  a 
leaf.  They  may  have  a  slightly  concentric  appearance.  The  col- 
onies rapidly  increase  in  size  and  become  more  and  more  opaque.  The 
gelatin  is  not  liquefied. 

Gelatin  Punctures. — Development  in  gelatin  punctures  occurs 
upon  the  surface,  and  also  in  the  needle's  track,  causing  the  forma- 
tion of  a  nail-like  growth.  The  head  of  the  nail  may  reach  the  walls 
of  the  test-tube.  No  gas  is  formed  in  ordinary  gelatin,  but  should 
any  dextrose  be  present,  sufficient  gas-production  may  occur  to 
break  up  the  medium.  The  gelatin  may  become  slightly  clouded 
but  is  not  liquefied. 

Agar-agar. — Upon  agar-agar,  along  the  line  of  inoculation,  a  gray- 
ish-white, translucent,  smeary  growth,  devoid  of  any  characteristics, 
takes  place.  The  entire  surface  of  the  culture-medium  is  never  cov- 
ered, the  growth  remaining  confined  to  the  inoculation  line,  except 


6i8  Bacillus  Coli 

where  the  moisture  of  condensation  allows  it  to  spread  out  at  the 
bottom.  Kruse  says  that  crystals  may  form  in  old  cultures. 

Bouillon. — Bouillon  is  densely  clouded  by  the  growth  of  the  bac- 
teria, a  delicate  pellicle  at  times  forming  upon  the  surface.  There  is 
usually  considerable  sediment  in  the  culture. 

Potato. — Upon  potato  the  growth  is  luxuriant.  The  bacillus 
forms  a  yellowish-brown,  glistening  layer  spreading  from  the  line 
of  inoculation  over  about  one-half  to  two-thirds  of  the  potato. 
The  color  varies  considerably,  sometimes  being  pale,  sometimes  quite 
brown,  sometimes  greenish.  It  cannot,  therefore,  be  taken  as  a  char- 
acteristic of  much  importance.  The  growth  on  potato  may  be  almost 
invisible. 

Milk. — In  milk  rapid  coagulation  and  acidulation  occur,  with  the 
evolution  of  gas.  The  culture  gives  off  a  fecal  odor.  Litmus  added 
to  the  culture-media  is  first  reddened,  then  decolorized  by  the  bacilli. 

Vital  Resistance. — It  is  quite  resistant  to  antiseptics  and  germi- 
cides, and  grows  in  culture-media  containing  from  0.1-0.2  per  cent, 
of  carbolic  acid.  It  is,  however,  easily  killed  by  heat,  and  is 
destroyed  by  exposure  to  6o°C.  for  ten  minutes. 

Metabolic  Products. — Wurtz  found  that  Bacillus  coli  produced 
ammonia  in  culture-media  free  from  sugar,  and  thus  caused  an  in- 
tense alkaline  reaction  in  the  culture-media.  The  cultures  usually 
give  off  an  odor  that  varies  somewhat,  but  is,  as  a  rule,  unpleasant. 

Nitrates  are  reduced  to  nitrites  by  the  growth  of  the  bacillus. 

In  bouillon  containing  i  per  cent,  of  dextrose,  lactose,  levulose, 
galactose,  and  mannite,  the  colon  bacillus  splits  up  the  sugar,  lib- 

H        2 

crating   CO2   and  H,   the  gas  formula  being    -—-  =  -.     This  gas 

\^\j%      i 

formula  is  very  constant  for  the  micro-organisms  of  the  colon  group 
and  forms  one  of  their  most  important  differential  characteristics. 
In  calculating  the  gas  formula  vWinslow  has  shown  that  some  care 
ought  to  be  taken  to  do  it  at  the  appropriate  time.  According  to  his 

H     2 
observations  the  -=^~  •  -  formula  only  obtains  between  the  twenty- 

{s\J2     1 

fourth  and  forty-eighth  hours.  Before  this  period  the  H,  which  is 
first  formed,  preponderates;  after  it  the  COz  may  preponderate. 
In  sugar-containing  bouillon,  acetic,  •  lactic,  and  formic  acids  are 
produced.  It  does  not  ferment  saccharose.  When  a  similar  bacillus 
is  found  to  ferment  saccharose  it  is  best  regarded  as  a  subspecies 
or  separate  type,  for  which  Dunham  has  introduced  the  name 
Bacillus  coli  communior. 

The  bacillus  requires  very  little  nutriment.  It  grows  in  Uschin- 
sky's  asparagin  solution,  and  is  frequently  found  living  in  river  and 
well  waters. 

Indol  is  formed  in  both  bouillon  and  peptone  solutions,  but  phenol 
is  not  produced.  The  presence  of  indol  is  best  determined  by  Sal- 
kowski's  method  (q.v.). 


Bacilli  Resembling  the  Typhoid  Bacillus  619 

Toxic  Products. — Vaughan  and  Cooley*  have  shown  that  the 
toxin  of  the  colon  bacillus  is  contained  in  the  germ-cell  and  under 
ordinary  conditions  does  not  diffuse  from  it  into  the  culture-medium. 
The  toxin  may  be  heated  in  water  to  a  very  high  temperature  without 
injuring  its  poisonous  nature.  They  have  devised  an  apparatus  in 
which  enormous  cultures  can  be  prepared  and  the  bacteria  pulver- 
ized, f  Of  such  a  preparation  0.0002  gram  will  kill  a  2oo-gram 
guinea-pig. 

Pathogenesis. — The  bacillus  begins  to  penetrate  the  intestinal 
tissues  almost  immediately  after  death,  and  is  the  most  frequent 
contaminating  micro-organism  met  with  in  cultures  made  at  autopsy. 
It  may  spread  by  direct  continuity  of  tissue,  or  via  the  blood-vessels. 

Although  under  normal  conditions  a  saprophyte,  the  colon  bacillus 
is  not  infrequently  found  in  the  pus  in  suppurations  connected  with 
the  intestines — as,  for  example,  appendicitis — and  sometimes  in 
suppurations  remote  from  them. 

In  intestinal  diseases,  such  as  typhoid,  cholera,  and  dysentery, 
the  bacillus  not  only  seems  to  acquire  an  unusual  degree  of  virulence, 
but  because  of  the  existing  denudation  of  mucous  surfaces,  etc.,  finds 
it  easy  to  enter  the  general  system,  with  the  formation  of  remote 
secondary  suppurative  lesions  in  which  it  is  the  essential  factor. 
When  absorbed  from  the  intestine,  it  frequently  enters  the  kidney 
and  is  excreted  with  the  urine,  causing,  incidentally,  local  inflamma- 
tory areas  in  the  kidney,  and  occasionally  cystitis.  A  case  of  ure- 
thritis  is  reported  to  have  been  caused  by  it. 

In  infants  cholera  infantum  may  not  infrequently  be  caused  by 
the  colon  bacillus,  though  sometimes  in  this  disease  other  bacteria 
play  an  important  role  (B.  dysenteriae?). 

The  bile-ducts  are  sometimes  invaded  by  the  bacillus,  which  may 
lead  to  inflammation,  obstruction,  suppuration,  or  calculus  formation. 

The  colon  bacillus  has  also  been  met  with  in  puerperal  fever, 
Winckel's  disease  of  the  newborn, {  endocarditis,  meningitis,  liver- 
abscess,  bronchopneumonia,  pleuritis,  chronic  tonsillitis,  urethritis, 
and  arthritis. 

An  interesting  summary  of  the  pathogenic  effect  of  Bacillus  coli 
can  be  found  in  Rolleston's  paper  in  the  "  British  Medical  Journal"  for 
Nov.  4,  1911,  p.  1186. 

In  a  certain  number  of  cases  general  hemic  infection  may  be  caused 
by  Bacillus  coli.  In  1909  Jacob§  published  an  analysis  of  39  such 
cases,  and  in  i9ioDraper||  increased  the  number  1043.  Wiens**also 
reported  6  cases  and  Maherff  i  case,  so  that  the  total  now  stands  50. 

*  "Jour.  Amer.  Med.  Assoc.,"  1901;  "American  Medicine,"  1901. 

f  "Trans.  Assoc.  Amer.  Phys.,"  1901. 

j  "  Kamen-Ziegler's  Beitrage,"  1896,  14. 

§  "Deutsch.  Archiv.  f.  Klin.  Med.,"  1909,  xcvn,  303. 

I!  "Bull,  of  the  Ayer  Clin.  Lab.  of  the  Penna.  Hosp.,"  1910,  No.  6,  p.  21. 

"Munch,  med.  Woch.,"  1909,  LVI,  962. 
ft  "Med.  Record,"  1909,  LXXV,  482. 


620  Bacillus  Coli 

Virulence. — It  is  a  question  whether  the  colon  bacillus  is  always 
virulent,  or  whether  it  becomes  so  under  abnormal  conditions. 
Klencki*  found  it  very  virulent  in  the  ileum,  and  less  so  in  the  colon 
and  jejunum  of  dogs.  He  also  found  that  the  virulence  was  greatly 
increased  in  a  strangulated  portion  of  intestine.  Dreyfusj  found 
that  the  colon  bacillus  as  it  occurs  in  normal  feces  is  not  virulent. 
Most  experimenters  believe  that  pathologic  conditions,  such  as 
disease  of  the  intestine,  strangulation  of  the  intestine,  etc.,  increase 
its  virulence. 

Frequent  transplantation  lessens  the  virulence  of  the  bacillus; 
passage  through  animals  increases  it. 

It  has  been  observed  that  cultures  of  the  bacillus  obtained  from 
cases  of  cholera,  cholera  nostras,  and  other  intestinal  diseases  are 
more  pathogenic  than  those  obtained  from  normal  feces  or  from  pus. 

Adelaide  Ward  Peckham,J  in  an  elaborate  study  of  the  "Influence 
of  Environment  on  the  Colon  Bacillus,"  concludes  that  while  the  con- 
ditions of  nutrition  and  development  in  the  intestine  seem  to  be  most 
favorable,  the  colon  bacillus  is  ordinarily  not  virulent.  She  says: 

"Its  first  force  is  spent  upon  the  process  of  fermentation,  and  as  long  as  oppor- 
tunities exist  for  the  exercise  of  this  function  the  affinities  of  this  organism  appear 
to  be  strongest  in  this  direction. 

"Moreover,  the  contents  of  the  intestine  remain  acid  until  they  reach  the 
neighborhood  of  the  colon,  and  by  that  time  the  tryptic  peptones  have  been 
formed  and  absorbed  to  a  great  extent. 

"During  the  process  of  inflammation  in  the  digestive  tract  a  very  different 
condition  may  exist.  The  peptic  and  tryptic  enzymes  may  be  partially  sup- 
pressed. Fermentation  of  carbohydrates  and  proteid  foods  then  begins  in  the 
stomach,  and  continues  after  the  mass  of  fo6d  is  passed  on  into  the  intestine. 
The  colon  bacillus  cannot,  therefore,  spend  its  force  upon  fermentation  of  sugars, 
because  they  are  already  broken  up  and  an  alkaline  fermentation  of  the  proteids 
is  in  progress.  It  also  cannot  form  peptones  from  the  original  proteids,  for  it  does 
not  possess  this  property,  and  unless  trypsin  is  present  it  must  be  dependent  upon 
the  proteolytic  activity  of  other  bacteria  for  a  suitable  form  of  proteid  food. 
Perhaps  these  bacteria  form  an  attmminate  molecule  which,  like  leucin  and 
tyrosin,  cannot  be  broken  up  into  indol,  and  thus  there  might  be  caused  an  im- 
portant modification  of  the  metabolism  of  the  colon  bacillus,  which  might  have 
either  an  immediate  or  remote  influence  upon  its  acquisition  of  disease-producing 
properties,  for  our  own  experiments  indicate  that  the  power  to  form  indol,  and 
the  actual  forming  of  it,  are  to  some  extent  an  indication  of  the  possession  of 
pathogenesis." 

For  the  laboratory  animals  the  colon  bacillus  is  pathogenic  in 
varying  degree.  Intraperitoneal  injections  into  mice  .cause  death 
in  from  one  to  eight  days  if  the  culture  be  virulent.  Guinea-pigs 
and  rabbits  also  succumb  to.  intraperitoneal  and  intravenous  in- 
jection. Subcutaneous  injections  are  of  less  effect,  and  in  rabbits 
produce  abscesses  only. 

When  injected  into  the  abdominal  cavity,  the  bacilli  set  up  a  sero- 
fibrinous  or  purulent  peritonitis,  and  are  very  numerous  in  the  ab- 
dominal fluids. 

*  "Ann.  de  1'Inst.  Pasteur,"  1895,  No.  9. 
•  "Centralbl.  f.  Bakt.,"  etc.,  xvi,  p.  581. 
I  "Journal  of  Experimental  Medicine,"  Sept.,  1897,  vol.  n,  No.  4,  p.  549. 


Bacilli  Resembling  the  Typhoid  Bacillus  621 

Cumston,*  from  a  careful  study  of  13  cases  of  summer  infantile  diar- 
rheas, comes  to  the  following  conclusions: 

Bacterium  coli  seems  to  be  the  pathogenic  agent  of  the  greater  number  of  sum- 
mer infantile  diarrheas. 

The  organism  is  often  associated  with  Streptococcus  pyogenes. 

The  virulence,  more  considerable  than  in  the  intestine  of  a  healthy  child,  is 
almost  always  in  direct  relation  to  the  condition  of  the  child  at  the  time  the  cul- 
ture is  taken,  and  does  not  appear  to  be  proportionate  to  the  ulterior  gravity  of 
the  case. 

The  mobility  of  Bacterium  coli  is,  in  general,  proportionate  to  its  virulence. 
The  jumping  movement,  nevertheless,  does  not  correspond  to  an  exalted  virulence 
in  comparison  with  the  cases  in  which  the  mobility  was  very  considerable,  without 
presenting  these  jumping  movements. 

The  virulence  of  Bacterium  coli  found  in  the  blood  and  other  organs  is  identical 
with  that  of  Bacterium  coli  taken  from  the  intestines  of  the  same  individual. 

Lesage,f  in  studying  the  enteritis  of  infants,  found  that  in  40  out 
of  50  cases  depending  upon  Bacillus  coli  the  blood  of  the  patient 
agglutinated  the  cultures  obtained,  not  only  from  his  own  stools,  but 
from  those  of  all  the  other  cases.  From  this  uniformity  of  action 
Lesage  suggests  that  the  colon  bacilli  in  these  cases  are  all  of  the  same 
species. 

The  agglutinating  reaction  occurs  only  in  the  early  stages  and  acute 
forms  of  the  disease. 

Immunization. — It  is  not  difficult  to  immunize  an  animal  against 
the  colon  bacillus.  Loffler  and  Abel  immunized  dogs  by  progressively 
increased  subcutaneous  doses  of  live  bacteria,  grown  in  solid  cul- 
ture and  suspended  in  water.  The  injections  at  first  produced  hard 
swellings.  The  blood  of  the  immunized  animals  possessed  an  active 
bactericidal  effect  upon  the  colon  bacteria.  The  serum  was  not  in 
the  correct  sense  antitoxic. 

Differential  Diagnosis. — This  problem  is  considered  at  greater 
length  under  the  heading  "Cultural  Differentiation  of  the  Bacillus 
Typhosus"  (q.v.).  For  the  recognition  of  the  colon  bacillus  the  most 
important  points  are  the  motility,  the  indol-formation,  the  milk- 
coagulation,  and  the  active  gas-production.  As,  however,  most  of 
these  features  are  shared  by  other  bacteria  to  a  greater  or  less  degree, 
•  the  most  accurate  differential  point  is  the  immunity  reaction  with  the 
serum  of  an  immunized  animal,  which  protects  susceptible  animals 
from  the  effects  of  inoculation,  and  produces  a  similar  agglutinative 
reaction  to  that  observed  in  connection  with  the  blood  and  serum  of 
typhoid  patients,  convalescents,  and  immunized  animals. 

The  fact  that,  with  rare  exceptions,  the  typhoid  serum  produces  a 
specific  reaction  with  the  typhoid  bacillus,  and  the  colon  serum  with 
the  colon  bacillus,  should  be  the  most  important  evidence  that  they 
are  entirely  different  species. 

What  is  commonly  known  as  Bacillus  coli  communis  is,  no  doubt, 

*  "International  Medical  Magazine,"  Feb.,  1897. 
f  "La  Semaine  Me"dicale,"  Oct.  20,  1897. 


622  Bacillus  Coli 

not  a  single  species,  but  a  group  of  bacilli  too  similar  to  be  differen- 
tiated into  groups,  types,  or  families  by  our  present  methods. 

In  order  to  establish  a  type  species  of  Bacillus  coli  communis, 
Smith*  says : 

"I  would  suggest  that  those  forms  be  regarded  as  true  to  this  species  which  grow 
on  gelatin  in  the  form  of  delicate  bluish  or  more  opaque,  whitish  expansions  with 
irregular  margin,  which  are  actively  motile  when  examined  in  the  hanging  drop 
from  young  surface  colonies  taken  from  gelatin  plates,  which  coagulate  milk 
within  a  few  days;  grow  upon  potato,  either  as  a  rich  pale  or  brownish- yellow 
deposit,  or  merely  as  a  glistening,  barely  recgonizable  layer,  and  which  give  a 
distinct  indol  reaction.  Their  behavior  in  the  fermentation-tube  must  conform 
to  the  following  scheme : 

"  Variety  a: 

"One  per  cent,  dextrose-bouillon  (at  37°C.).  Total  gas  approximately  %\ 
H  :  C(>2  =  approximately  2:1;  reaction  strongly  acid. 

"One  per  cent,  lactose-bouillon:  as  in  dextrose-bouillon  (with  slight  varia- 
tions). 

"One  per  cent,  saccharose-bouillon;  gas-production  slower  than  the  preceding, 
lasting  from  seven  to  fourteen  days.  Total  gas  about  %;  H  :  CO2  =  nearly 
3 : 2.  The  final  reaction  in  the  bulb  may  be  slightly  acid  or  alkaline,  according  to 
the  rate  of  gas-production  (B.  coli  communior,  Dunham). 

"Variety  ft: 

"The  same  in  all  respects,  excepting  as  to  its  behavior  in  saccharose-bouillon; 
neither  gas  nor  acids  are  formed  in  it." 

DIFFERENTIAL  CHARACTERISTICS 

TYPHOID  BACILLUS  COLON  BACILLUS 

Bacilli  usually  slender.  Bacilli  a  little  thicker  and  shorter. 

Flagella  numerous  (10-20),  long,  and  Flagella  fewer  (8-10)  (peritricha). 

wavy  (peritricha). 

Growth  not  very  rapid,  not  particu-  Growth    rapid    and    luxuriant.     This 

larly  luxuriant.  character  is  by  no  means  constant. 

Upon  Eisner's,  Hiss',  Piorkowski's,  and  Upon  Eisner's,  Hiss',  Piorkowski's,  and 

other  media  gives  characteristic  ap-  other     media     gives     characteristic 

pearances.  appearances. 

Upon  fresh  acid  potato  the  so-called  Upon  potato  a  brownish-yellow  distinct 

"invisible  growth"  formerly  thougTit  pellicle. 

to  be  differential. 

Acid-production  in  whey  not  exceeding  Acid-production  well  marked 

3  per  cent.     Sometimes  slight  in  or-  throughout. 

dinary    media,    and    succeeded    by 

alkali-production. 

Grows  in  media  containing  sugars  with-  Fermentation  with  gas-production  well 

out  producing  any  gas.  marked     in     solutions      containing 

dextrose,  lactose,  etc.,  the  usual  for- 
mula being  H    :   CO2  =  2:1. 

Produces  no  indol.  Indol-production  marked. 

Growth    in    milk    unaccompanied    by  Milk  coagulated. 

coagulation. 

Gives  the  Widal  reaction  with  the  ser-  Does  not  react  with  typhoid  blood. 

um  of  typhoid  blood. 

Colon  Bacillus  in  Drinking  Water. — Much  importance  attaches  to 
the  presence  or  absence  of  colon  bacilli  in  judging  the  potability  of 
drinking  waters. 

*  "Amer.  Jour.  Med.  Sci.,"  1895,  no,  p.  287. 


Bacilli  Resembling  the  Typhoid  Bacillus  623 

It  is  a  speculation  whether  the  colon  bacilli  were  originally  micro- 
organisms of  the  soil  that  accidentally  found  their  way  into  the  con- 
genial environment  of  the  intestine  and  there  took  up  permanent 
residence,  or  whether  they  have  always  been  intestinal  parasites  and 
have  been  discharged  with  the  excrement  of  animals  until  the  soil 
has  become  generally  infected  with  them.  However  this  may  be, 
they  are  at  present  found  in  the  intestinal  canals  of  all  animals,  and 
in  pretty  much  all  soils,  their  number  being  greatest  in  manured  soils. 
From  the  soil  it  is  inevitable  that  the  organisms  shall  pass  into  the 
surface  waters,  which  with  few  exceptions  will  be  found  to  contain 
them.  The  numbers,  however,  can  be  made  use  of  to  indicate  the 
quality  of  the  water,  a  few  organisms  indicating  that  the  water  is 
pure,  many  that  it  is  freely  mixed  with  surface  washings. 

As  sewage  contains  as  many  as  1,000,000  colon  bacilli  per  cubic 
centimeter  and  pure  water  very  often  o  per  cubic  centimeter  (only 
i  cc.  being  examined  at  a  time),  the  number  of  bacilli  per  cubic 
centimeter  can  be  expressed  as  indicating  the  amount  of  sewage 
pollution.  The  number  of  colon  bacilli  in  the  water  is,  therefore,  of 
importance  in  determining  its  potability,  and  in  cases  in  which 
the  quality  of  the  water  is  doubtful,  should  always  be  employed. 
There  is  no  infallible  criterion  for  judging  the  quality  of  water,  but 
most  American  bacteriologists  are  in  accord  in  concluding  that  when 
the  repeated  examination  of  i  cc.  samples  shows  the  presence  of 
numerous  colon  bacilli,  the  water  is  seriously  polluted  and  doubtfully 
potable,  but  when  samples  of  i  cc.  are  without  colon  bacilli  or  contain 
very  few,  the  water  is  safe. 

Another  important  matter  in  regard  to  the  colon  bacillus  in  water  is 
the  presence  or  absence  of  certain  characters  by  which  one  can  judge 
how  recently  it  has  ended  its  intestinal  parasitism  and  taken  up  a 
saprophytic  life.  The  chief  of  these  characters  is  the  ability  to  fer- 
ment lactose.  Only  recently  isolated  organisms  manifest  this  fer- 
mentative power  in  the  laboratory,  so  that  when  organisms  capable 
of  fermenting  lactose  are  found,  one  can  suppose  that  they  result 
from  recent  sewage  pollution. 

Many  media  have  been  recommended  for  the  rapid  detection  of  the 
colon  bacilli  in  water,  the  favorite  at  the  present  time  probably  be- 
ing the  litmus-lactose-agar  plate  (q.v.)  of  Wlirtz.  *  This  depends  upon 
the  fermentative  and  acid-producing  power  of  the  bacillus,  which  is 
shown  through  the  presence  of  red  colonies  (acid  producers)  on  the 
elsewhere  blue  plate.  These  red  colonies  are  then  fished  up  and  trans- 
planted to  appropriate  media  for  further  study. 

Kline  |  substitutes  lactose  for  the  glucose  in  this  medium,  pointing 
out  that  by  so  doing  one  at  once  differentiates  between  typical  colon 
bacilli  which  ferment  lactose  and  atypical  varieties  which  do  not. 

*  "Archiv.  de  m6d.  Experimentale,"  1892,  iv,  p.  85. 
t  "British  Medical  Journal,"  Oct.  27,  1906,  p.  1090. 


624  Bacillus  Faecalis 

Other  media  and  methods  useful  in  studying  the  colon  bacilli  are 
also  discussed  in  the  chapter  upon  Typhoid  Fever  (q.v.). 

BACILLUS  ENTERITIDIS  (GARTNER) 

General  Characteristics. — A  motile,  flagellated,  non-sporogenous,  non-chro- 
mogenic,  non-liquefying,  aerogenic,  aerobic  and  optionally  anaerobic,  pathogenic 
bacillus  staining  by  the  ordinary  methods,  but  not  by  Gram's  method. 

This  bacillus  was  first  cultivated  by  A.  Gartner*  from  the  flesh  of  a  cow 
slaughtered  because  of  an  intestinal  disease,  and  from  the  spleen  of  a  man 
poisoned  by  eating  meat  obtained  from  it.  The  bacillus  was  subsequently  found 
by  Karlinski  and  Lubarsch  in  other  cases  of  meat-poisoning. 

Morphology. — The  bacillus  closely  resembles  Bacillus  coli  communis.  It 
is  short  and  thick,  is  surrounded  by  a  slight  capsule,  is  actively  motile,  and  has 
flagella. 

Staining. — It  stains  irregularly  with  the  ordinary  solutions,  but  not  by  Gram's 
method.  It  has  no  spores. 

Cultivation. — Upon  gelatin  plates  it  forms  round,  pale  gray,  translucent  col- 
onies. It  does  not  liquefy  the  gelatin.  The  deep  colonies  are  brown  and  spheric. 
The  growth  on  agar-agar  is  similar  to  that  of  the  colon  bacillus.  The  organism 
produces  no  indol,  coagulates  milk  in  a  few  days,  and  reduces  litmus.  Its  fer- 
mentative powers  have  not  been  sufficiently  studied,  but  it  is  known  to  ferment 
dextrose  media.  Upon  potato  it  forms  a  yellowish-white,  shining  layer. 

Pathogenesis. — The  bacillus  is  pathogenic  for  mice,  guinea-pigs,  pigeons, 
lambs,  and  kids,  but  not  for  dogs,  cats,  rats,  or  sparrows.  The  infection  may  be 
fatal  for  mice  and  guinea-pigs,  whether  given  subcutaneously,  intraperitoneally, 
or  by  the  mouth. 

Lesions. — The  bacilli  are  found  scattered  throughout  the  organs  in  small 
groups,  resembling  those  of  the  typhoid  bacillus. 

At  the  autopsy  a  marked  enteritis  and  swelling  of  the  lymphatic  follicles  and 
patches,  with  occasional  hemorrhages,  are  found.  The  bacilli  occur  in  the  intes- 
tinal contents.  The  spleen  is  somewhat  enlarged. 

The  bacillus  is  differentiated  from  the  colon  bacillus  chiefly  by  the  absence  of 
indol-production,  by  its  ability  to  produce  infection  when  ingested,  and  by  the 
fact  that  it  elaborates  a  toxic  substance  capable  of  producing  symptoms  similar 
to  those  seen  in  the  infection. 

It  may  be  distinguished  from  Bacillus  lactis  aerogenes  by  its  motility.  It 
closely  resembles  certain  water  bacteria;  but  its  pathogenesis  can  be  made  use  of 
for  assisting  in  its  differentiation  in  doubtful  cases. 

V 

BACILLUS  FAECALIS  ALKALIGENES  (PETRUSCHKY) 

General  Characteristics. — A  motile,  flagellated,  non-sporogenous,  non-liquefy- 
ing, non-chromogenic,  non-aerogenic,  aerobic  and  optionally  anaerobic,  non- 
pathogenic  bacillus  of  the  intestine,  staining  by  ordinary  methods,  but  not  by 
Gram's  method. 

This  bacillus  has  occasionally  been  isolated  by  Petruschkyf  and  others  from 
feces.  It  closely  resembles  the  typhoid  bacillus,  being  short,  stout,  with  round 
ends,  forming  no  spores,  staining  with  the  usual  dyes,  but  not  by  Gram's  method, 
being  actively  motile,  and  having  numerous  flagella.  It  does  not  liquefy  gelatin, 
does  not  coagulate  milk,  produce  gas,  or  form  indol.  Its  pathogenic  powers  for 
the  lower  animals  are  similar  to  those  of  the  typhoid  bacillus. 

It  grows  more  luxuriantly  than  the  typhoid  bacillus  upon  potato,  producing  a 
brown  color,  and  generates  a  strong  alkali  when  grown  in  litmus-whey.  Its  cul- 
tures are  not  agglutinated  by  the  typhoid  serums. 

"Korrespond.  d.  allg.  arztl.  Ver.  von  Thuring,"  1888,  9. 
t  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  XDC,  187. 


Bacilli  Resembling  the  Typhoid  Bacillus  625 

BACILLUS  PSITTACOSIS  (NOCARD) 

General  Characteristics. — A  motile,  flagellated,  non-sporogenous,  aerobic, 
optionally  anaerobic,  non-chromogenic,  aerogenic,  pathogenic,  non-liquefying 
bacillus,  staining  by  the  ordinary  methods,  but  not  by  Gram's  method. 

This  micro-organism  was  discovered  by  Nocard,*  who  first  observed  it  in  1892 
in  certain  cases  of  psittacosis,  or  epidemic  pneumonia,  traceable  to  infection  from 
diseased  parrots.  The  original  paper  contained  an  excellent  account  of  the  spe- 
cific organism. 

The  subsequent  work  of  Gilbert  and  Fournierf  shows  the  specificity  of  the 
micro-organism  to  be  quite  well  established  and  Nocard 's  characterizations 
accurate. 

Morphology. — The  bacillus  is  short,  stout,  rounded  at  the  ends,  and  actively 
motile.  It  is  provided  with  flagella,  but  forms  no  spores.  It  resembles  the 
typhoid  and  the  colon  bacilli  and  is  evidently  a  form  intermediate  between 
the  two. 

Isolation. — Gilbert  and  Fournier  succeeded  in  isolating  it  from  the  blood  of  a 
patient  dead  of  psittacosis,  and  from  parrots,  by  the  use  of  lactose-litmus  agar. 
The  organism  does  not  alter  the  litmus,  and  if  a  small  percentage  of  carbolic  acid 
be  added  to  the  culture-media,  it  grows  as  does  the  typhoid  bacillus. 

Cultivation. — The  colonies,  agar-agar  and  gelatin  cultures,  closely  resemble 
those  of  the  typhoid  fever  organism.  Upon  potato  it  more  closely  resembles  the 
colon  bacillus.  Bouillon  becomes  clouded. 

Metabolic  Products. — In  bouillon  containing  sugars  the  micro-organism  is 
found  to  ferment  dextrose,  but  not  lactose.  Milk  is  not  coagulated  and  not 
acidulated.  No  indol  is  formed. 

Pathogenesis. — Bacillus  psittacosis  can  be  immediately  differentiated  from  the 
typhoid  and  colon  bacilli  by  its  peculiar  pathogenesis.  It  is  extremely  virulent 
for  parrots,  producing  a  fatal  infection  in  a  short  time.  White  and  gray  mice  and 
pigeons  are  equally  susceptible.  Ten  drops  of  a  bouillon  culture  injected  in  the 
ear- vein  of  a  rabbit  kill  it  in  from  twelve  to  eighteen  hours.  Guinea-pigs  are 
more  resistant.  Subcutaneous  injection  of  dogs  produces  a  hard,  painful  swelling, 
which  persists  for  a  short  time  and  then  disappears  without  suppuration.  It  is 
also  infectious  for  man,  a  number  of  epidemics  of  peculiar  pneumonia,  character- 
ized by  the  presence  of  the  bacillus  in  the  blood,  traceable  to  diseased  parrots, 
having  been  reported. 

Differentiation. — Bacillus  psittacosis  can  best  be  differentiated  from  the  ty- 
phoid and  the  colon  bacilli  and  others  of  the  same  group  by  its  pathogenesis  and  by 
the  reaction  of  agglutination.  Typhoid  immune  serum  produces  some  small 
agglutinations,  but  a  comparison  between  these  and  the  agglutinations  formed  by 
cultures  of  the  typhoid  bacillus  shows  immediately  that  the  micro-organisms  are 
dissimilar.  Differentiation  is  best  made  out  when  the  prepared  hanging-drop 
specimens  of  serums  and  cultures  are  kept  for  some  hours  in  an  incubating  oven. 
It  is  not  known  whether  the  bacillus  is  peculiar  to  the  intestines  of  parrots,  invad- 
ing their  tissues  when  they  become  ill,  or  whether  it  is  a  purely  pathogenic  micro- 
organism found  only  in  psittacosis. 

BACILLUS  SUIPESTIFER  (SALMON  AND  SMITH) 

General  Characteristics. — An  actively  motile,  flagellated,  non-sporogenous, 
non-chromogenic,  non-liquefying,  aerobic  and  optionally  anaerobic,  aerogenic 
bacillus  pathogenic  for  hogs  and  other  animals.  It  stains  by  the  ordinary 
methods,  but  not  by  Gram's  method.  It  ferments  dextrose,  lactose,  and  sucrose, 
but  does  not  form  indol  or  coagulate  or  acidulate  milk. 

Hog-cholera,  or  "pig  typhoid,"  as  the  English  call  it,  is  a  common  epidemic 
disease  of  swine,  which  at  times  kills  90  per  cent,  of  the  infected  animals,  and  thus 
causes  immense  losses  to  breeders.  Salmon  estimates  that  the  annual  losses 

*  "  Seance  du  Conseil  d'hygiene  publique  et  Salubrite  du  Departement  de  la 
Seine,"  March  24,  1893. 

t"Comptes  rendu  de  la  Societe  de  Biologic,"  1896;  "La  Presse  medicale," 
Jan.  16,  1897. 


626  Bacillus  Suipestifer 

from  this  disease  in  the  United  States  range  from  $10,000,000  to  $25,000,000. 
For  years  it  was  thought  to  be  caused  by  the  Bacillus  suipestifer,  but 
DeSchweinitz  and  Dorset*  were  able  to  transmit  the  disease  from  one  hog  to 
another  in  certain  of  the  body  fluids  that  had  been  passed  through  the  finest  por- 
celain niters  and  were  shown  by  inoculation  and  cultivation  to  be  free  of  bacilli. 
It  therefore  depends  upon  a  filterable  and  unknown  virus. 

This  observation  was  received  with  approval  by  those  who  had  any  experience 
with  the  effect  of  hog-cholera  bacilli  upon  hogs,  all  of  whom  must  have  observed 
that  though  infection  with  the  bacilli  occasionally  caused  the  death  of  an  animal, 
the  dead  animal  usually  did  not  show  the  typical  lesions  of  the  disease  and  never 
infected  other  animals  with  which  it  was  kept.  The  papers  upon  the  subject  by 
Dorset,  Bolton,  and  McBrydef  and  by  Dorset,  McBryde,  and  NilesJ  are  worth 
reading. 

These  investigations  entirely  changed  our  ideas  of  the  importance  of  the  hog- 
cholera  bacillus,  whose  relation  to  the  disease  now  comes  to  resemble  that  of 
Bacillus  icteroides  to  yellow  fever. 

The  bacillus  of  hog-cholera  was  first  found  by  Salmon  and  Smith,  §  but  was  for 
a  long  time  confused  with  the  bacillus  of  "  swine-plague,"  which  it  closely  resem- 
bles, and  in  association  with  which  it  frequently  occurs.  It  is  a  member  of  the 
group  of  bacteria  to  which  Bacillus  icteroides  and  B.  typhi  murium  belong.  The 
organism  was  secured  by  Smith  from  the  spleens  of  more  than  500  hogs.  It  occurs 
in  the  blood  and  in  all  the  organs,  and  has  also  been  cultivated  from  the  urine. 

Morphology. — The  organisms  appear  as  short  rods  with  rounded  ends,  1.2  to 
l  .5  n  long  and  0.6  to  0.7  M  in  breadth.  They  are  actively  motile  and  possess  long 
flagella  (peritrichia),  easily  demonstrable  by  the  usual  methods  of  staining.  No 
spore  production  has  been  observed.  In  general  the  bacillus  resembles  that  of 
typhoid  fever.  It  stains  readily  by  the  ordinary  methods,  but  not  by  Gram's 
method. 

Cultivation. — No  trouble  is  experienced  in  cultivating  the  bacilli,  which  grow 
well  in  all  the  media  under  aerobic  and  anaerobic  conditions. 

Colonies. — Upon  gelatin  plates  the  colonies  become  visible  in  from  twenty 
four  to  forty-eight  hours,  the  deeper  ones  appearing  spheric  with  sharply  define- 
borders.  The  surfaces  are  brown  by  reflected  light,  and  without  markingsd 
They  are  rarely  larger  than  0.5  mm.  in  diameter  and  are  homogeneous  through-, 
out.  The  superficial  colonies  have  little  tendency  to  spread  upon  the  gelatin. 
They  rarely  reach  a  greater  diameter  than  2  mm.  The  gelatin  is  not  liquefied. 

Upon  agar-agar  they  attain  a  diameter  of  4  mm.  and  have  a  gray,  translucent 
appearance  with  polished  surface.  They  are  round  and  slightly  arched. 

Gelatin. — In  gelatin  punctures  the  growth  takes  the  form  of  a  nail  with  a  flat 
head.  There  is  nothing  characteristic  about  it.  The  medium  is  not  liquefied. 

Agar-agar. — Linear  cultures  upon  agar-agar  present  a  translucent,  circum- 
scribed, grayish,  smeary  layer  without  characteristic  appearances. 

Potato. — Upon  potato  a  yellowish  coating  is  formed,  especially  when  the  culture 
is  kept  in  the  thermostat. 

Bouillon. — Bouillon  made  with  or  without  peptone  is  clouded  in  twenty-four 
hours.  When  the  culture  is  allowed  to  stand  for  a  couple  of  weeks  without  being 
disturbed,  a  thin  surface  growth  can  be  observed. 

Milk  is  an  excellent  culture-medium,  but  is  not  visibly  changed  by  the  growth 
of  these  bacteria.  Its  reaction  remains  alkaline. 

Vital  Resistance.— The  bacillus  is  hardy.  Smith  found  it  vital  after  being  dry 
for  four  months.  It  ordinarily  dies  sooner,  however,  and  difficulty  may  be  ex- 
perienced in  keeping  it  in  the  laboratory  for  any  length  of  time  unless  frequently 
transplanted.  The  thermal  death-point  is  S4°C.,  maintained  for  sixty  minutes. 

Metabolic  Products. — Gas  Production.— The  hog-cholera  bacillus  is  a  copious 
gas-producer,  capable  of  breaking  up  dextrose  and  lactose  into  CO2,  H,  and  an 

*  "  Circular  No.  41  of  Bureau  of  Animal  Industry,"  U.  S.  Dept.  of  Agriculture, 
Washington,  D.  C. 

t"Bull.  No.  72  of  Bureau  of  Animal  Industry,"  U.  S.  Dept.  Agriculture, 
Washington,  D.  C.,  1905. 

t"Bull.  No.  102  of  Bureau  of  Animal  Industry,"  U.  S.  Dept.  Agriculture, 
Washington,  D.  C.,  Jan.  18,  1908. 

§  "Reports  of  the  Bureau  of  Animal  Industry,"  1885-91;  and  "Centralbl.  f. 
Bakt.  u.  Parasitenk.,"  March  2,  1891,  Bd.  ix,  Nos.  8,  9,  and  10. 


Bacilli  Resembling  the  Typhoid  Bacillus  627 

acid,  which,  formed  late,  eventually  checks  its  further  development.  It  does  not 
ferment  saccharose. 

Indol. — No  indol  and  no  phenol  are  formed  in  the  culture-media. 

Toxin. — In  pure  cultures  of  the  hog-cholera  bacillus  Novy*  found  a  poisonous 
base  with  the  probable  composition  CieH^e^,  which  he  gave  the  provisional 
name  "  susotoxin."  In  doses  of  100  mg.  the  hydrochlorid  of  this  base  causes  con- 
vulsive tremors  and  death  within  one  and  one-half  hours  in  white  rats.  He  has 
also  obtained  a  poisonous  protein  of  which  50  mg.  were  fatal  for  white  rats,  and 
which  immunized  them  against  highly  virulent  hog-cholera  organisms  when 
administered  by  repeated  subcutaneous  injection. 

De  Schweinitzf  has  also  separated  a  slightly  poisonous  base  which  he  calls 
"sucholotoxin,"  and  a  poisonous  protein  that  crystallizes  in  white,  translucent 
plates  when  dried  over  sulphuric  acid  in  vacua,  forms  needle-like  crystals  with 
platinic  chlorid,  and  was  classed  among  the  albumoses. 

Pathogenesis. — The  bacillus  is  disappointing  in  its  effects  upon  hogs.  When 
it  is  subcutaneously  or  intravenously  introduced  into  such  animals  or  fed  to  them, 
they  sometimes  show  no  signs  of  disease;  sometimes  show  fever  and  depression, 
but  rarely  sicken  enough  to  die.  Animals  thus  made  ill  do  not  communicate 
hog  cholera  to  others. 

Smith  found  that  0.75  cc.  of  a  bouillon  culture  injected  into  the  breast  mus- 
cles of  pigeons  would  kill  them. 

In  Smith's  experiments  one  four-millionth  of  a  cubic  centimeter  of  a  bouillon 
culture  injected  subcutaneously  into  a  rabbit  was  sufficient  to  cause  its  death. 
The  temperature  abruptly  rises  2°  to  3°C.,  and  remains  high  until  death.  Sub- 
cutaneous injection  of  larger  quantities  may  kill  in  five  days.  Injected  intra- 
venously in  small  doses  the  bacillus  may  kill  rabbits  in  forty-eight  hours. 

Agglutination. — PitfieldJ  found  that  after  a  single  injection  of  a  killed  bouillon 
culture  of  the  bacillus  into  a  horse,  the  serum,  which  originally  had  very  slight 
agglutinative  power,  showed  a  decided  increase.  If  the  horse  be  immunized  to 
large  doses  of  such  sterile  cultures,  the  serum  becomes  so  active  that  with  a 
dilution  of  i :  10,000  a  typical  agglutination  occurs  in  sixty  minutes. 

McClintock,  Boxmeyer  and  Siffer§  found  that  the  serum  of  normal  hogs 
agglutinates  strains  of  ordinary  hog-cholera  bacilli  in  dilutions  occasionally  as 
high  as  1 1250  and  consider  reaction  in  a  dilution  of  less  than  1 1300  without  diag- 
nostic value. 

BACILLUS  ICTEROIDES  (SANARELLI) 

General  Characteristics. — An  actively  motile,  flagellated,  non-sporogenous, 
non-liquefying,  non-chromogenic,  aerogenic,  aerobic  and  optionally  anaerobic, 
pathogenic  bacillus  which  stains  by  the  ordinary  method,  but  not  by  Gram's 
method.  It  produces  indol,  but  does  not  coagulate  milk. 

Sanarelli  regarded  this  bacillus  as  the  specific  organism  of  yellow  fever.  He 
found  it  in  n  autopsies  upon  yellow  fever  cases,  but  always  in  association  with 
streptococci,  colon  bacilli,  proteus,  and  other  organisms.  It  is  found  in  the 
blood  and  tissues,  and  not  in  the  gastro-intestinal  tract,  and  isolation  of  the 
organism  was  possible  in  only  58  per  cent,  of  the  cases,  and  only  in  rare  instances 
was  accomplished  during  life. 

Distribution. — By  suitable  methods  it  can  be  found  in  the  organs  of  yellow 
fever  cadavers,  usually  aggregated  in  small  groups,  in  the  capillaries  of  the  liver, 
kidneys,  and  other  organs.  The  best  method  of  demonstration  is  to  keep  a  frag- 
ment of  liver,  obtained  from  a  body  soon  after  death,  in  the  incubator  at  37°C.  for 
twelve  hours,  and  allow  the  bacteria  to  multiply  in  the  tissue  before  examination. 

Morphology. — The  bacillus  presents  nothing  morphologically  characteristic. 
It  is  a  small  pleomorphous  bacillus  with  rounded  ends,  usually  joined  in  pairs.  It 
is  2  to  4  ju  in  length,  and,  as  a  rule,  two  or  three  times  longer  than  broad.  It  is 
actively  motile  and  has  flagella.  It  does  not  form  spores. 

Staining. — It  stains  by  the  usual  methods,  but  not  by  Gram's  method. 

*  "Medical  News,"  1900,  p.  231. 

t  "Medical  News,"  1900,  p.  237. 

J  "  Microscopical  Bulletin,"  1897,  p.  35. 

§  "Jour,  of  Infectious  Diseases,"  March  i,  1905,  vol.  n,  No.  2,  p.  351. 


628 


Bacillus  Murium 


Cultivation. — The  bacillus  can  be  grown  upon  the  usual  media.  It  grows  read- 
ily at  ordinary  room  temperatures,  but  best  at  37°C. 

Colonies. — Upon  gelatin  plates  it  forms  rounded,  transparent,  granular  col- 
onies, which  during  the  first  three  or  four  days  somewhat  resemble  leukocytes. 
The  granular  appearance  becomes  continuously  more  marked,  and  usually  an 
opaque  central  or  peripheral  nucleus  is  seen.  In  time  the  entire  colony  becomes 
opaque,  but  does  not  liquefy  gelatin. 

Gelatin. — Stroke  cultures  on  obliquely  solidified  gelatin  show  brilliant,  opaque, 
white  colonies  resembling  drops  of  milk.     The  medium  is  not  liquefied. 
Bouillon. — In  bouillon  it  develops  slowly,  without  either  pellicle  or  flocculi. 
Agar-agar. — The  culture  upon  agar-agar  is  said  to  be  characteristic. 

The  peculiar  and  characteristic  appearances  of  the 
colonies  do  not  develop  if  grown  at  37°C.;  but  at  20°  to 
22°C.  the  colonies  appear  rounded,  whitish,  opaque,  and 
prominent,  like  drops  of  milk.  This  appearance  of  the 
colonies  also  shows  well  if  the  cultures  are  kept  for  ,the 
first  twelve  to  sixteen  hours  at  37°C.,  and  afterward  at 
the  room  temperature,  when  the  colonies  will  show  a  flat 
central  nucleus  transparent  and  bluish,  surrounded  by  a 
prominent  and  opaque  zone,  the  whole  resembling  a  drop 
of  sealing-wax.  Sanarelli  refers  to  this  appearance  as  con- 
stituting the  chief  diagnostic  feature  of  Bacillus  icteroides. 
It  can  be  observed  in  twenty-four  hours. 

Blood-serum. — Upon  blood-serum  the  growth  is  very 
meager. 

Potato. — The  growth  upon  potato  corresponds  with  that 
of  the  bacillus  of  typhoid  fever. 

Vital  Resistance. — It  strongly  resists  drying,  but  dies 
when  exposed  in  cultures  to  a  temperature  of  60°  C.  for  a 
few  minutes,  and  is  killed  in  seven  hours  by  the  solar  rays. 
It  can  live  for  a  considerable  time  in  sea-water. 

Metabolism. — The  bacillus  is  an  optional  anaerobe.  It 
slowly  ferments  dextrose,  lactose,  and  saccharose,  form- 
ing gas  only  in  dextrose  solutions  in  which  there  are  no 
other  sugars.  It  does  not  coagulate  milk.  In  the  cultures 
a  small  amount  of  indol  is  formed. 

Pathogenesis. — The  bacillus  is  pathogenic  for  the  do- 
mestic animals,  all  mammals  seeming  to  be  more  or  less 
sensitive  to  it.  Birds  are  often  immune.  White  mice  are 
killed  in  five  days,  guinea-pigs  in  from  eight  to  twelve 
days,  rabbits  in  from  four  to  five  days,  by  virulent  cultures. 
The  morbid  changes  present  include  splenic  tumor,  hyper- 
trophy of  the  thymus,  and  adenitis.  In  the  rabbit  there 
are,  in  addition,  nephritis,  enteritis,  albuminuria,  hemo- 
globinuria,  and  hemorrhages  into  the  body  cavities. 

Sanarelli  states  that  the  dog  is  the  most  susceptible  animal. 
When  this  animal  is  injected  intravenously,  symptoms  ap- 
pear almost  immediately  and  recall  the  clinical  and  ana- 
tomic features  of  yellow  fever  in  man.  The  most  prominent 
symptom  in  the  dog  is  vomiting,  which  begins  directly  after 
the  penetration  of  the"  virus  into  the  blood,  and  continues 
for  a  long  time.  Hemorrhages  appear  after  the  vomiting, 
the  urine  is  scanty  and  albuminous,  or  is  suppressed  shortly 
before  death.  Grave  jaundice  was  once  observed. 

BACILLUS  TYPHI  MURIUM  (LOFFLER) 

General  Characteristics. — A  motile,  flagellated,  non-sporogenous,  non-liquefy- 
ing, non-chromogenic,  non-aerogenic,  aerobic  and  optionally  anaerobic  bacillus, 
pathogenic  for  mice  and  other  small  animals,  staining  by  the  ordinary  methods, 
but  not  by  Gram's  method.  It  acidulates  but  does  not  coagulate  milk. 

Bacillus  typhi  murium  was  discovered  by  Lorner*  in  1889,  when  it  created  havoc 
among  the  mice  in  his  laboratory  at  Greifswald. 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  xi,  p.  129. 


Fig.  256. — Cul- 
ture of  Bacillus 
icteroides  on  agar 
(Sanarelli). 


Bacilli  Resembling  the  Typhoid  Bacillus 


629 


Morphology. — The  organism  bears  a  close  resemblance  to  that  of  typhoid  fever, 
sometimes  appearing  short,  sometimes  long  and  flexible.  There  are  many  long 
and  curly  flagella  with  peritrichial  arrangement,  and  the  organism  is  actively 
motile.  It  does  not  produce  spores. 

Staining. — It  stains  with  the  ordinary  dyes,  but  rather  better  with  Lomer's 
alkaline  methylene  blue,  not  by  Gram's  method. 

Isolation. — The  bacilli  were  first  isolated  from  the  blood  of  dead  mice. 

Cultivation. — Their  cultivation  presents  no  difficulties. 

Colonies. — Upon  gelatin  plates  the  deep  colonies  are  at  first  round,  slightly 
granular,  transparent,  and  grayish.  Later  they  become  yellowish  brown  and 
granular.  Superficial  colonies  are  similar  to  those  of  the  typhoid  bacillus. 

Gelatin. — In  gelatin  punctures  there  is  no  liquefaction.  The  growth  takes 
place  principally  upon  the  surface,  where  a  grayish- white  mass  slowly  forms,  and 
together  with  the  growth  in  the  puncture  suggests  a  large  flat-headed  nail.- 

Agar-agar. — Upon  agar-agar  a  grayish-white  growth  devoid  of  peculiarities 
occurs. 

Potato. — Upon  potato  a  rather  thin  whitish  growth  may  be  observed  after  a 
few  days. 

Milk. — The  bacillus  grows  well  in  milk,  causing  acid  reaction,  without  coagu- 
lation. 


Fig.  257. — Bacillus  typhi  murium  (Migula). 

Bouillon. — In  bouillon  it  produces  clouding.  There  is  no  fermentation  of 
saccharose,  dextrose,  lactose,  or  levulose. 

Pathogenesis. — The  organism  is  pathogenic  for  mice  of  all  kinds,  which  suc- 
cumb in  from  one  to  two  days  when  inoculated  subcutaneously,  and  in  from  eight 
to  twelve  days  when  fed  upon  material  containing  the  bacillus.  The  bacilli 
multiply  rapidly  in  the  blood-  and  lymph-channels,  and  cause  death  from 
septicemia. 

Lofner  expressed  the  opinion  that  this  bacillus  might  be  of  use  in  ridding 
infested  premises  of  mice,  and  its  use  for  this  purpose  has  been  satisfactory  in 
many  places.  He  has  succeeded  in  ridding  fields  so  infested  with  mice  as  to  be 
useless  for  agricultural  purposes,  by  saturating  bread  with  bouillon  cultures  of  the 
bacillus  and  distributing  it  near  their  holes.  The  bacilli  not  only  killed  the  mice 
that  had  eaten  the  bread,  but  also  infected  others  that  ate  their  dead  bodies,  the 
extermination  progressing  until  scarcely  a  mouse  remained. 

In  discussing  the  practical  employment  of  this  bacillus  for  the  satisfactory  de- 
struction of  field-mice,  B runner*  calls  attention  to  certain  conditions  that  are 
requisite:  (i)  It  is  necessary,  first  of  all,  to  attack  extensive  areas  of  the  invaded 
territory,  and  not  to  attempt  to  destroy  the  mice  of  a  small  field  into  which  an 
*  "Centralbl.  f.  Bakt,"  etc.,  Jan.  19,  1898,  Bd.  xxm,  No.  2,  p.  68. 


630  Bacillus  Murium 

indefinite  number  of  fresh  animals  may  immediately  come  from  surrounding 
fields.  The  country  people,  who  are  the  sufferers,  should  combine  their  efforts  so  as 
to  extend  the  benefits  widely.  (2)  The  preparation  of  the  cultures  is  a  matter  of 
importance.  Agar-agar  cultures  are  most  readily  transportable.  They  are 
broken  up  in  water,  well  stirred,  and  the  liquid  poured  upon  a  large  number  of 
small  pieces  of  broken  bread.  These  are  then  distributed  over  the  ground  with 
care,  being  dropped  into  the  fresh  mouse-holes,  and  pushed  sufficiently  far  in  to 
escape  the  effects  of  sunlight  upon  the  bacilli.  Attention  should  be  paid  to  holes 
in  walls,  under  railway  tracks,  etc.,  and  other  places  where  mice  live  in  greater 
freedom  from  disturbance  than  in  the  fields.  (3)  The  destruction  of  the  mice 
should  be  attempted  only  at  a  time  of  the  year  when  their  natural  food  is  not 
plenty.  By  observing  these  precautions  the  mice  can  be  eradicated  in  from  eight 
to  twelve  days.  In  the  course  of  two  years  no  less  than  250,000  cultures  were 
distributed  from  the  Bacteriological  Laboratory  of  the  Tierarznei  Institut  in 
Vienna,  for  the  purpose  of  destroying  field-mice. 

The  bacilli  are  not  pathogenic  for  animals,  such  as  the  fox,  weasel,  ferret,  etc., 
that  feed  upon  the  mice,  do  not  affect  man  in  any  way,  and  so  seem  to  occupy  a 
useful  place  in  agriculture  by  destroying  the  little  but  almost  invincible  enemies 
of  the  grain. 

A  similar  organism,  secured  from  an  epidemic  among  field-mice  and  greatly 
increased  in  virulence  by  artificial  manipulation,  has  been  recommended  by 
Danysz*  for  the  destruction  of  rats.  When  subjected  to  a  thorough  study  by 
Rosenauf  this  organism  was  found  to  be  identical  with  Bacillus  typhi  murium. 
It  is,  however,  too  uncertain  in  action  to  be  relied  upon  for  the  destruction  of  rats 
in  plague-threatened  cities  for  which  it  was  suggested. 

*  "Ann.  de  ITnst.  Pasteur,"  April,  1900. 

t  "  Bulletin  No.  5  of  the  Hygienic  Laboratory  of  the  U.  S.  Marine  Hospital 
Service,"  Washington,  D.  C.,  1901. 


CHAPTER  XXVIII 
DYSENTERY 

DYSENTERY  is  an  acute,  subacute,  or  chronic,  infectious  colitis, 
usually  characterized  by  an  acute  onset,  mild  fever,  pain  in  the  abdo- 
men, rectal  tenesmus,  and  the  passage  of  frequent,  usually  small, 
mucous  and  bloody  evacuations  from  the  rectum. 

The  disease  was  known  to  the  ancients.  It  was  probably  dysen- 
tery that  is  meant  by  "  emerods  "  in  describing  an  epidemic  that  took 
place  among  the  people  of  Israel  during  the  time  of  the  Judges.  Hip- 
pocrates differentiated  between  diarrhea  and  dysentery. 

Sporadic  cases  of  the  disease  occur  in  almost  all  countries,  the 
number  of  such  increasing  as  the  equator  is  approached.  In  addition 
to  these  sporadic  cases  epidemics  not  infrequently  appear.  Though 
such  may  break  out  at  any  time  in  towns  or  cities,  they  are  more  apt 
to  occur  when  unusual  activities  of  the  people  are  in  progress.  The 
most  frequent  of  these  is  military,  and  armies  are  apt  to  be  the  great- 
est sufferers.  The  incidence  of  dysentery  in  the  Federal  Army  dur- 
ing the  War  of  the  Rebellion  was  appalling.  Woodward*  states 
that  there  were  259,071  cases  of  acute  and  28,451  cases  of  chronic 
dysentery. 

Endemics  also  occur  from  time  to  time  and  assume  devastating 
proportions,  as  in  Japan,  where  between  1878  and  1899  there  were 
1,136,096  cases,  with  275,308  deaths — a  mortality  of  25.23  per  cent.f 
Osier  quotes  Macgregor  as  saying:  "In  the  tropics  dysentery  is  a 
destructive  giant  compared  to  which  strong  drink  is  a  mere  phantom. 
It  is  one  of  the  great  camp  diseases  and  has  been  more  destructive 
to  armies  than  powder  and  shot." 

The  disease  early  came  under  the  observation  of  the  bacteriolo- 
gists, and  Klebs,  Ziegler,  Ogata,  Grigorjeff,  de  Silvestri,  Maggiora, 
Arnaud,  Celli  and  Fiocca,  Galli-Valerio,  Valagussa,  Deycke,  and 
others  published  descriptions  of  various  micro-organisms  isolated 
from  dysenteric  stools,  and  looked  upon  by  their  discoverers  as  its 
cause.  The  results  were,  however,  so  discordant  that  none  of 
the  described  micro-organisms  could  be  agreed  upon  as  the  excitant 
of  the  disease. 

In  1860  LamblJ  published  a  description  of  an  ameba  found  in  the 
human  intestine.  No  one  seemed  inclined  to  believe  that  it  might 
have  any  significance  until  much  later. 

In  1875  L6sch§  described  an  ameba  which  he  found  in  great  num- 

*  "  Medical  and  Surgical  History  of  the  War  of  the  Rebellion,"  Medical,  n. 

"Public  Health  Reports,"  Jan.  5,  1900,  xv,  No.  i. 
$  "  Aus.  d.  Franz  Joseph  Kinderspital  zur  Prague,"  1860,  i,  326. 
§  "Virchow's  Archives,"  1875,  Bd.  LXV. 

631 


632  Dysentery 

bers  in  the  colon  of  a  case  of  dysentery  occurring  in  St.  Petersburg. 
Not  much  notice  was  taken  of  his  paper  or  much  made  of  his  obser- 
vation until  eight  years  later,  when  Koch  and  Gaffky,*  in  studying 
the  cholera  in  Egypt,  also  observed  amebas  in  the  intestinal  dis- 
charges in  certain  cases,  and  Kartulisf  wrote  upon  the  "  Etiology  of 
the  Dysentery  in  Egypt,"  which  he  referred  to  them.  In  America 
the  study  of  these  amebas  was  quickly  taken  up.  Osier  {  dis- 
covered the  organisms  in  the  evacuations  of  a  case  of  dysentery 
contracted  by  a  patient  during  a  visit  to  Panama.  Councilman  and 
Lafleur§  wrote  a  fine  monograph  upon  "Amebic  Dysentery,"  while 
Quincke  and  Roos||  and  Kruse  and  Pasquale**  confirmed  the  ob- 
servations and  results  in  Europe. 

Thus  it  came  to  be  recognized  that  an  ameba  might  be  the  cause  of 
dysentery.  It  was  soon  pointed  out,  however,  that  there  were  cases 
of  dysentery  in  which  no  amebas  could  be  found  in  the  intestinal  dis- 
charges, or  in  which  they  were  so  few  that  it  seemed  impossible  that 
they  could  be  the  cause  of  the  disease.  This  was  particularly  im- 
pressive throughout  the  years  of  the  endemic  dysentery  in  Japan, 
already  referred  to.  Great  numbers  of  cases  occurred,  great  num- 
bers of  people  died,  no  amebas  were  found  to  account  for  the  disease. 
It  therefore  occurred  to  Kitasato  that  some  other  causal  agent  must 
be  looked  for,  and  Shiga  took  up  the  problem,  which  was  a  difficult 
one,  and  might  not  have  been  solved  had  he  not  made  use  of  a  then 
new  means  of  investigation,  viz.,  the  phenomenon  of  agglutination. 
By  studying  such  bacteria  as  could  be  cultivated  from  the  intes- 
tinal discharges,  with  particular  reference  to  the  agglutinating 
effect  of  the  blood  of  dysenteric  patients  upon  them,  Shiga  f|  suc- 
ceeded in  discovering  a  new  micro-organism  which  he  called  Bacillus 
dysenteriae.  Two  years  afterward  KruseJI  investigated  an  outbreak 
of  dysentery  in  an  industrial  section  of  Westphalia  and  found  the 
same  bacillus  and  Flexner§§  showed  it  to  be  present  in  the  epidemic 
dysentery  of  the  Philippine  Islands. 

Thus  through  the  discovery  of  Shiga  it  became  evident  that 
there  are  two  forms  of  dysentery,  one  amebic  the  other  bacillary. 
Both  occur  sporadically  and  endemically  in  the  tropics  and  in  tem- 
perate climates,  and  both  may  occur  epidemically,  though  of  the 
two  the  bacillary  form  is  the  more  liable  to  do  so.  Of  the  chronic 
cases  of  dysentery  90  per  cent,  are  amebic. 

""Bericht  uber  die  Erforschung  der  Cholera,"  1883;  "Arbeiten  aus  dem 
Kaiserl.     Gesundheitsamte.,"  in,  65. 
t  "Virchow's  Archives,"  1886,  cv. 
j  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1890,  vn,  736. 
§  "Johns  Hopkins  Hospital  Reports,"  1891,  n. 
"Berliner  klin.  Wochenschrift,"  1893. 
1  "Zeitschrift  f.  Hygiene,"  etc.,  1894,  xvi. 
"Gentralbl.  f.  Bakt.  u.  Parasitenk.,"  1898,  xxrv,  817. 
tt  "Deutsche  med.  Wochenschrift,"  1900,  No.  40. 
§§  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1900,  xxvm,  No.  19. 


Amebic  Dysentery 


I.  AMEBIC  DYSENTERY 


633 


AMOEBA  COLI  (LOSCH,  1875);  AMOEBA  DYSENTERIC  (COUNCILMAN 

AND  LAFLEUR,  1893);  ENTAMOEBA  HISTOLYTICA  (SCHAU- 

DINN,   1903) 

As  has  been  shown,  amebas  were  first  found  in  the  human  in- 
testine by  Lambl;  in  dysentery,  by  Losch,  Koch,  Gaffky,  Kartulis, 
Osier,  Councilman  and  Lafleur,  and  many  others.  The  welcome 
finally  accorded  to  the  organisms  as  excitants  of  dysentery  was 
sufficiently  enthusiastic  to  compensate  for  the  neglect  of  a  quarter 
of  a  century. 

Celli  and  Fiocca*  were  the  first  to  study  the  amebas  system- 
atically and  to  cultivate  them  upon  artificial  media.  Councilman 
and  Lafleur  pointed  out  that  there  were  two  varieties  of  amebas 
which  they  called  Amceba  coli  and  Amceba  dysenteriae.  The 
former  was  supposed  to  be  a  harmless  commensal,  the  latter  a 
pathogenic  organism  and  the  cause  of  dysentery.  As,  however. 


Fig.  258. — Amoeba  coli  in  intestinal  mucus,  with  blood-corpuscles  and 
bacteria  (Losch). 

Losch  had  called  the  organism  found  in  dysentery  the  Amoeba 
coli,  Stiles  declared  the  nomenclature  faulty,  and  pointed  out  that 
Amceba  coli,  variety  dysenteriae,  must  be  the  name  of  the  patho- 
genic form.  Schaudinn  f  reviewed  the  subject  and  grouped  all  of 
the  intestinal  amebas  under  the  following: 

I.  Chlamydophrys  stercorea  (Cienkowsky). 
II.  Amceba  coli  rhizopodia. 

1.  Entamceba  coli  (Losch)  (Schaudinn). 

2.  Entamceba  histolytica  (Schaudinn). 
To  these  has  been  since  added  in  1907: 

Entamceba  tetragena  (Viereck). 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1894,  xv,  470. 

f  "Arbeiten  aus  d.  Kaiserl.  Gesundheitsamte.,"  1903,  xix,  No.  3. 


634  Dysentery 

1.  Entamoeba  Coli  (Losch,  1875). — This  organism  seems  to  be  a 
harmless  commensal,  living  in  the  intestines  of  man,  many  domestic, 
and  many  wild  animals.     It  may  be  abundant  when  the  reaction 
of  the  intestinal  contents  is  neutral  or  alkaline.     It  usually  measures 
between  10  and  20/1  in  diameter  when  free,  but  when  encysted  from 
15  to  50  /*.     It  is  spheroidal  when  not  in  motion,  and  under  these 
conditions  it  is  difficult  to  differentiate  endoplasm  and  ectoplasm. 
The  ameboid  movement  is  sluggish  and  the  pseudopods  are  rather 
short,  broad,  and  blunt.     As  they  are  protruded  the  clear  ectoplasm 
becomes  visible.     The  organism  has  a  grayish  color,  a  finely  granular 
cytoplasm,  and  usually  only  a  single  vacuole.     The  nucleus  is  usually 
fairly  well  defined  and  spherical,  and,  in  addition  to  the  chromatin, 
contains  several  nucleoli.     When  stained  with  polychrome  methy- 
lene-blue  the  ectoplasm  stains  blue;  the  endoplasm,  violet;  and  the 
nucleus,  red. 

Reproduction  usually  takes  place  by  simple  division,  but  a  form 
of  autogamous  sporulation  also  takes  place,  the  organism  first  be- 
coming encysted,  the  nucleus  dividing  into  eight  segments,  and 
the  whole  process  eventuating  in  the  formation  of  eight  young 
organisms. 

This  ameba  is  easily  cultivated  upon  artificial  media  according 
to  methods  to  be  described  below. 

It  is  not  pathogenic,  and  all  attempts  to  make  it  damage  the 
intestines  of  experiment  animals  have  failed. 

2.  Entamoeba  Histolytica  (Schaudinn*). — -This  is  now  recognized 
as  the  organism  seen  by  Losch,  Koch,  Kartulis,  Councilman  and 
Lafleur,  and  accepted  as  the  cause  of  the  amebic  form  of  dysentery. 
It  is  found  in  all  parts  of  the  world,  but  more  frequently  in  tropical 
than  colder  climates,  and  is  present  only  in  the  intestines  of  those 
suffering  from  dysentery.     It  is  usually  present  in  great  numbers 
so  that  its  discovery  in  the  evaluations  is  easy. 

Morphology. — It  is  usually  considerably  larger  than  Entamoeba 
coli  and  varies  in  diameter  up  to  50  ju.  When  at  rest  it  is  spherical, 
when  active  it  is  very  irregular.  Its  movement  is  active  and  the 
pseudopodia  are  larger  and  more  numerous  than  in  the  other  species. 
The  differentiation  of  ectoplasm  and  endoplasm  is  usually  distinct. 
The  former  is  hyaline,  the  latter  granular.  The  protoplasm  has  a 
greenish  or  yellowish  color.  The  nucleus  is  small,  not  very  distinct. 
There  are  numerous  vacuoles.  In  the  intestinal  evacuations  of 
dysentery  its  protoplasm  commonly  contains  many  red  blood- 
corpuscles,  upon  which  the  organism  seems  to  feed. 

Staining. — When  stained  with  polychrome  methylene-blue  the 
ectoplasm  stains  more  deeply  than  the  endoplasm.  The  nucleus 
contains  relatively  little  chromatin. 

Reproduction. — -Multiplication  takes  place  by  binary  division 
after  karyokinesis  and  by  encystment  and  sporulation.  The  sporula- 
*  "Arbeiten  a.  d.  k.  k.  Gesundheitsamt.,"  1903,  xix,  547. 


Amebic  Dysentery 


635 


tion  is  quite  different  from  that  seen  in  Entamoeba  coli,  and  only 
takes  place  when  conditions  are  unfavorable  to  continued  division. 
It  is  accomplished  by  a  peculiar  nuclear  budding,  by  which  chromatin 
granules  or  chronidia  are  pushed  out  from  the  nucleus  toward  the 
ectoplasm,  where  they  develop  into  new  nuclei,  about  which  the 
cytoplasm  collects  until  a  distinct  bud  is  formed  and  cast  off  as  a 
small  but  distinct  new  organism — a.  spore  or  bud.  These  when 
separated  are  round  or  oval,  measure  3  to  6  ^u  in  diameter,  and  are 


Fig.  259. — Reproductive  cycle  of  parasitic,  ameba  (Wenyon).  The  small 
circle  indicated  by  i,  2,  3,  3'  and  3"  indicated  multiplication  by  schizogony  or 
binary  division.  The  large  circle  indicated  by  1-12,  thesporogeny  or  sexual  cycle. 
The  ameba  having  arrived  at  its  full  size  (3)  becomes  encysted  (4).  The  nu- 
cleus then  divides  into  two  (5),  each  half  expels  a  small  fragment  of  nuclear 
material  (6),  and  when  this  has  been  effected,  they  conjugate  (7)  forming 
a  synkaryon.  The  synkaryon  then  divides  into  two,  into  four,  and  then 
generally  into  eight  (8-9-10-11-12)  when  the  cyst  ruptures,  the  spores  are  liber- 
ated (i)  and  both  cycles  are  again  started. 

surrounded  by  a  yellowish  envelope,  which  resists  drying  and  the 
penetration  of  stains  and  chemicals. 

Craig  gives  a  tabulation  of  the  differential  features  of  Entamoeba 
coli,  Entamoeba  histolytica,  and  Entamoeba  tetragena  (Me  infra). 

3.  Entamoeba  Tetragena  (Viereck*). — This  organism  resembles 
Entamoeba  histolytica  more  than  Amoeba  coli,  but  differs  from  it 
in  the  mode  of  reproduction,  the  sporocysts  containing  four  instead 
of  eight  spores. 

*  "Archiv.  f.  Schiffs.  u.  Tropenhygiene,"  1907,  n,  i. 


636  Dysentery 

Relationship  of  the  Organisms. — In  recent  years  (1910-1915)  much 
morphological  and  experimental  study  of  these  amcebas  has  been 
conducted  with  results  that  are  given  in  full,  together  with  the 
literature,  in  a  paper  "The  Identity  of  Entamceba  Histolytica  and 
Entamceba  Tetragena,  with  Observations  upon  the  Morphology  and 
Life  Cycle  of  Entamceba  Histolytica"  by  Charles  F.  Craig.*  The 
results  of  his  studies,  as  set  forth  in  the  paper,  go  to  show  that 
Schaudinn  was  in  error  in  regard  to  the  developmental  cycle  of 
Entamceba  histolytica,  that  what  he  supposed  to  be  its  sole  method 
of  reproduction,  is  only  that  means  that  preponderates  during  the 
period  of  its  greatest  activity;  that  as  the  acme  of  the  dysenteric 
disease  is  passed  and  the  process  of  repair  sets  in,  the  other  mode  of 
reproduction  characteristics  of  Entamceba  tetragena  is  observed, 
and  that  the  two  species  Entamceba  histolytica  and  Entamceba 
tetragena  are  one.  There  is,  therefore,  to  all  appearances,  and 
according  to  the  best  information  available  at  present,  only  one 
pathogenic  intestinal  amceba,  the  Entamceba  histolytica.  The 
same  conclusions  have  also  been  arrived  at  by  Darling,  f 

With  regard  to  Entamceba  coli,  opinion  as  to  its  non-pathogenic 
disposition  is  much  less  certain  than  a  few  years  ago.  Williams  and 
Calkins J  close  their  excellent  paper  upon  "Cultural  Amceba;  a 
Study  in  Variation"  with  the  statement  that  "it  is  unwise  for  any- 
one at  present  to  be  too  positive  in  regard  to  the  distinctive  features 
of  Entamceba  coli,  E.  tetragena  and  E.  histolytica,  or  any  of  the 
Entamceba  groups.  There  may  be  in  man,  three  or  more,  or  two 
(as  Hartmann,  Whitman,  Walker  and  Craig  now  think)  or  possibly 
only  one  species  of  ameba  manifesting  different  forms  under  different 
conditions." 

Isolation  and  Cultivation. — Many  experimenters  have  made 
more  or  less  successful  attempts  to  cultivate  amebas.  Musgrave  and 
Clegg,§  whose  interesting  paper  the  student  will  do  well  to  read,  and 
in  which  he  will  find  a  complete  review  of  all  antecedent  work,  were 
able  to  cultivate  a  considerable  variety  of  amebas  upon  agar-agar 
made  of: 

Agar 20 .  o          grams 

Sodium  chlorid o .  3-0 . 5 

Extract  of  beef o .  3-0 . 5       " 

Water : 1000 .  o             cc. 

Prepare  as  ordinary  culture  agar,  and  render  i  per  cent,  alkaline  to  phenol- 
phthalein.  The  finished  medium  is  poured  into  Petri  dishes.  To  obtain  the  greatest 
number  of  most  active  amebas  the  patient  should  be  given  a  dose  of  a  saline 
purgative,  and  the  fluid  evacuation  resulting  from  its  action  employed  for 
inoculating  the  media.  The  cultures  are,  naturally,  not  pure;  they  contain 
various  amebas  and  numerous  bacteria. 

*  "Jour.  Infectious  Diseases,"  1913,  xm,  30. 

f  "Trans,  of  the  Fifteenth  International  Congress  on  Hygiene  and  Dermo- 
graphy,"  Washington,  D.  C.,  Sept.,  1912. 

$  "Jour.  Med.  Research,"  1913-1914,  xxiv,  43. 

§  "  Department  of  the  Interior,  Bureau  of  Government  Laboratories,  Biological 
Laboratory,"  Manila,  Oct.,  1904,  No.  8. 


Amebic  Dysentery  637 

To  isolate  and  cultivate  a  single  kind  of  ameba  Musgrave  and 
Clegg  have  recommended  an  ingenious  technic. 

A  plate  is  selected  upon  which  the  desired  amebas  are  so  widely  separated  from 
one  another  that  not  more  than  one  is  in  a  microscopic  field  of  a  low-power 
objective.  The  microscope  used  should  have  a  double  or  triple  nose-piece. 
With  a  low-power  (Zeiss  A  A)  objective,  a  well-isolated  organism  is  brought  to  the 
center  of  the  field.  The  lens  is  then  swung  out  and  a  perfectly  clean  higher-power 
lens  (Zeiss  D  D)  swung  in  and  racked  down  until  it  touches  the  surface  of  the 
agar-agar,  when  it  is  quickly  elevated  again.  In  three  out  of  five  cases  the  ameba 
adheres  to  the  objective  and  is  so  picked  up.  Whether  it  has  done  so  or  not  can 
be  determined  by  swinging  in  the  low-power  lens  again  and  looking  for  the  organ- 
ism. If  it  has  disappeared,  it  is  attached  to  the  objective.  It  is  now  planted 
upon  a  fresh  plate  by  depressing  the  high-power  lens  until  it  touches  the  surface 
of  the  culture-medium,  when,  upon  elevating  it  again,  it  usually  leaves  the 
ameba  behind.  Observation  with  the  low  power  will  enable  one  to  determine 
whether  it  be  successfully  planted  or  not. 

Naturally  the  organisms  cannot  be  thus  transplanted  without  some  bacteria 
falling  upon  the  plate,  but  this  is  not  very  material,  for  in  the  first  place  they  do 
not  grow  very  rapidly  upon  the  medium  used  for  culture,  and  in  the  second,  they 
are  useful  for  the  nourishment  of  the  ameba,  which  is  holophagous,  and  cannot 
live  by  the  absorption  of  nutritious  fluids. 

Later  it  was  shown  by  Tsugitani*  that  killed  cultures  of  bacteria 
could  supply  the  necessary  nourishment.  All  cultures  of  amebas 
must  contain  some  kind  of  cells  upon  which  the  amebas  can  feed. 
When  planted  as  above  suggested  a  variety  of  organisms  grow,  and 
as  the  amebas  multiply  and  gradually  extend  over  the  plate,  their 
preference  for  one  or  other  of  the  associated  bacteria  may  be  deter- 
mined in  part  by  placing  a  drop  of  the  ameba  culture  upon  a  plate 
of  sterile  media,  and  then  with  the  platinum  wire,  dipped  in  a 
culture  of  the  bacteria,  and  drawing  concentric  circles  about  the  drop 
further  and  further  apart.  As  the  amebas  move  about  over  the 
plate,  passing  through  the  growing  circles  of  bacteria,  they  soon 
lose  the  miscellaneous  bacteria  and  come  to  contain  the  one  variety 
planted  with  them,  or  if  several  have  been  used  in  drawing  different 
circles,  that  one  which  they  prefer  to  feed  upon.  By  transplanting 
amebas  from  plate  to  plate  with  suitable  bacteria  or  other  cells  for 
them  to  feed  upon,  the  cultures  may  be  kept  growing  almost  in- 
definitely. 

Anna  Williamsf  has  been  able  to  grow  ameba  in  pure  culture 
without  bacteria,  either  dead  or  alive,  by  smearing  the  surface  of 
a  freshly  prepared  agar-agar  plate  with  a  fragment  of  freshly  re- 
moved rabbit's  or  guinea-pig's  brain,  kidney,  or  liver,  held  in  a  pair 
of  forceps.  The  ameba  gladly  take  up  and  live  upon  the  cells  left 
behind  upon  the  surface  of  the  agar. 

Vital  Resistance. — 'The  free  amebas  in  the  intestinal  discharges 
are  easily  destroyed  by  dilute  germicides  and  by  drying.  Encysted 
amebas  are,  however,  more  difficult  to  kill.  They  resist  drying 
well  and  also  resist  the  penetration  of  germicides.  Direct  sunlight 
inhibits  the  activities  of  the  organisms,  but  does  not  kill  them. 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Abt.  i,  xxrv,  666. 

t  "Journal  of  Medical  Research,"  Dec.,  1911,  xxv,  No.  2,  p.  263. 


Dysentery 


. 


^IG.    260. 


Amebic  Dysentery  639 

EXPLANATION  OF  FIG.  260 
(All  figures  drawn  by  Charles  F.  Craig,  M.  D.) 

I.  Upper  Group. — Entamceba  coli  stained  with  Giemsa  stain. 

Ay  B,  and  C.  Vegetative  organisms  showing  nuclear  membrane,  karyosome, 
and  collections  of  chromatin  upon  the  nuclear  membrane  and  within  the 
hyaloplasm.  Vacuoles  are  also  present. 

D.  An  organism  containing  a  protozoan  parasite  which  might  be  mistaken 

for  spores. 
H.  Division  of  nucleus  (primitive  mitosis). 

E.  Partially  divided  ameba  containing  two  nuclei. 

F.  G.  Ameba  resulting  from  simple  division. 

M.  Schizogony  of  Entamceba  coli.     Eight  daughter  nuclei  in  vegetative 

form. 

N.  Ameba  resulting  from  schizogony. 

/.  Earliest  stage  in  cyst  formation.  Cytoplasm  clear  of  foreign  bodies  and 
nucleus  showing  collection  of  chromidial  masses  upon  the  inner  side  of 
the  nuclear  membrane. 

p£,  L,  O,  P.  Two-  and  four-nucleated  stage  of  reproduction  within  the  cyst. 
\Q.  Encysted  form  containing  two  large  nuclei  and  a  mass  of  chromatin. 
I/?.  Fully  developed  cyst  of  Entamceba  coli  containing  eight  nuclei. 
Lower  Group. — Entamceba  coli,  fixed  in  sublimate  alcohol  and  stained  with  Dela- 
field's  hematoxylin.     Note  the  more  delicate  staining  of  the  nucleus  and 
the  greater  detail  obtained  with  this  method  of  staining. 
A,  B,  C.  Vegetative  amebae  showing  variations  in  the  structure  of  the 
nucleus. 

D.  An  organism  during  schizogony,  containing  eight  nuclei. 

E.  Mitotic  division  of  the  nucleus  as  observed  in  this  species. 

F.  A  fully  developed  cyst  of  Entamceba  coli  containing  eight  daughter 

nuclei. 

G.  The  four-nucleated  cystic  stage  of  Entamceba  coli  sometimes  mistaken 

for  the  cyst  of  Entamceba  tetragena. 

H.  Two-nucleated  cyst  of  Entamceba  coli. 

/.  Young  amebae  originating  from  the  cysts  of  Entamceba  coli. 

K.  Fully  developed  cyst  in  which  the  cystic  membrane  is  apparently  absent. 

L.  Degenerated  cyst  of  Entamceba  coli,  filled  with  vacuoles,  and  containing 

masses  of  chromatin.     No  nucleus  is  visible. 
II.  Entamoeba  histolytica  stained  with  Giemsa  stain. 

A.  Organism  showing  distinction  between  the  ectoplasm  and  endoplasm, 

nucleus  and  vacuole. 

B.  Organism  showing  vacuole  and  red  blood  corpuscle  and  nucleus  contain- 
ing minute  karyosome  and  chromatin  dots  in  the  hyaloplasm. 

C.  Organism  showing  nucleus  and  numerous  red  blood  corpuscles. 

D.  Organism  in  first  stage  of  nuclear  division,  showing  division  of  the  karyo- 

some and  minute  dots  of  chromatin  in  hyaloplasm. 

E.  Organism  showing  later  stage  of  nuclear  division,  the  polar  bodies  being 

connected  by  a  filament  of  chromatic  substance. 

F.  First  stage  of  formation  of  spore  cysts;  the  nucleus  distributing  chro- 

matin to  the  cytoplasm. 

G  to  7.  Stages  in  the  process  of  formation  of  spore  cysts,  the  chromatin  being 
distributed  to  the  cytoplasm  and  collected  in  threads  or  masses, 
while  the  nucleus  is  observed  as  a  flattened  body  crowded  against  the 
periphery  of  the  parasite. 

L.  Degenerated  parasite  containing  vacuoles  and  free  chromatin. 

K,  M,  N.  Entamceba  histolytica  in  the  final  stage  of  the  formation  of  spore 
cysts.  The  free  chromatin  has  collected  at  the  periphery,  and  sur- 
rounded by  a  small  amount  of  cytoplasm,  is  being  budded  off  from  the 
parent  organism. 

O.  Degenerated  organism  filled  with  vacuoles  and  free  from  chromatin. 
The  nucleus  stains  abnormally  and  there  is  no  distinction  between  the 
ectoplasm  and  endoplasm. 

P.  Entamceba  histolytica  filled  with  erthyrocy tes,  the  nucleus  being  crowded 
to  the  periphery  and  staining  abnormally  (Charles  F.  Craig,  M.  D., 
in  Journal  of  Medical  Research,  vol.  xxvi,  No.  i,  April,  1912). 


640 


Dysentery 


GROUP 


-OWER     GROUP 


* 


UPPER     GROUP 


mil 


mm. 

VMS 


J?t 


,«r  «s> 


> 


+ 

'4* 


LOWER     GROUP 


FIG.  261. 


Amebic  Dysentery  641 

EXPLANATION  OF  FIG.  261 

(All  figures  drawn  by  Charles  F.  Craig,  M.  D.  ) 

III.  Upper  Group. — Entamoeba  tetragena  fixed  in  sublimate  alcohol  and  stained 

with  Delafield's  hematoxylin.     Note  the  great  delicacy  of  the  stain- 
ing when  compared  with  the  staining  with  the  Giemsa  method. 

A.  A  vegetative  parasite  showing  three  erythrocytes  in  the  cytoplasm  and  a 

nucleus  in  which  the  nuclear  membrane,  and  the  karyosome  with  its 
centriole  are  shown. 

B.  A  vegetative  organism  showing  thick  nuclear  membrane  and  karyosome 

containing  a  centriole. 

C.  A  vegetative  parasite  containing  vacuoles  and  nucleus  showing  karyo- 

some containing  a  centriole  surrounded  by  an  unstained  area. 

D.  A  degenerative  form  filled  with  vacuoles  and  showing  abnormal  appear- 

ance of  the  nucleus. 

E.  Precystic  form  of  Entamceba  tetragena. 

G.  Another  precystic  form  which  is  more  typical  in  the  free  chromatin  in  the 
cytoplasm  is  visible.  The  form  E  would  probably  degenerate  before 
the  cyst  wall  was  fully  formed. 

F.  A  cystic  form  of  Entamoeba  tetragena  showing  two  chromatin  spindles  in 

the  cytoplasm  and  a  nucleus   having  a  centriole   surrounded  by  an 

unstained  area  and  a  definite  network  upon  which  are  arranged  dots  of 

chromatin. 
H.  An  encysted  form  showing  a  very  large  mass  of  chromatin  and  a  nucleus 

containing  a  karyosome  and  centriole. 

7.  Two-nucleated  cyst  of  Entamceba  tetragena  showing  mass  of  free  chroma- 
tin  and  the  morphology  of  the  nuclei  after  division. 
K.  Fully  developed  cyst  of  Entamceba  tetragena  containing  four  daughter 

nuclei  and  a  mass  of  chromatin. 
L.  Degenerated    form    of    Entamceba    tetragena    containing    some    free 

chromatin  and  a  nucleus  in  which  the  karyosome  stains  deeply  and 

nearly  fills  the  nucleus.     This  form  might  be  mistaken  for  a  free  living 

ameba. 
M.  Illustrating   the   typical   nuclear   structure   of   Entamoeba  tetragena. 

Note  the  large  karyosome  containing  a  centriole  surrounded  by  an 

unstained  area. 
Lower  Group. — Entamceba  histolytica  fixed  in  sublimate  alcohol  and  stained  with 

Delafield's  hematoxylin. 
A  and  B.  Vegetative  organisms  showing  vacuoles  and  typical  morphology  of 

the  nucleus.     No  distinction  between  the  endoplasm  and  ectoplasm. 

C.  Vegetative  form  of  Entamceba  histolytica  showing  the  type  of  mitosis 

during  simple  division. 

D.  First  step  in  the  formation  of  spore  cysts.     The  distribution  of  the  chro- 

matin by  the  nucleus  to  the  cytoplasm. 

E.  F  and  H.  Organisms  showing  chromidia  in   the  cytoplasm  arranged  in 

rods,  threads,  and  masses,  the  nucleus  being  flattened  out  against  the 
periphery  and  staining  poorly. 

G.  A  degenerative  form  of  Entamceba  histolytica  filled  with  vacuoles  and 

with  an  atypical  nucleus. 
/  and  K.  Budding  of  the  spore  cysts  from  the  periphery  of  Entamceba 

histolytica. 
L.  Illustrating  the  typical  nuclear  structure  of  Entamceba  histolytica. 

IV.  Upper  Group. — Entamceba  tetragena  stained  with  Giemsa  stain. 

A,  B,  C.  Vegetative  organisms.  Note  that  the  nuclear  membrane  and 
karyosome  stain  very  heavily  and  are  not  as  well  differentiated  as  in 
specimens  stained  with  hematoxylin. 

D.  Precystic  form  containing  masses  of  chromatin  in  the  cytoplasm. 

E.  Degenerative  form  containing  vacuoles,  masses  of  chromatin,  and  an 

atypically  stained  nucleus. 

F.  Two-nucleated  stage  of  the  cyst  of  Entamceba  tetragena,  showing  heavy 

staining  of  the  nuclear  membrane  and  karyosome.-    Two  masses  of 
chromatin  are  present. 
41 


642  Dysentery 

Losch  was  the  first  to  observe  that  quinin  was  destructive  to  in- 
testinal amebas,  and  his  observations  have  been  reviewed  by  many 
others.  Musgrave  and  Clegg  found  that  active  cultures  of  one 
ameba  were  killed  in  ten  minutes  by  a  1:2500  solution  of  quinin 
hydrochlorate.  The  exposed  organisms  quickly  encysted  themselves 
and  in  from  five  to  eight  minutes  many  of  them  had  broken  up  and 
disappeared.  After  ten  minutes  all  were  dead.  Cultures  of  another 
ameba  similarly  treated  gave  a  scanty  growth  after  ten  minutes. 

Vedder  found  that  emetin  would  kill  ameba  in  dilutions  up  to 
i :  100,000,  and  Rogers  has  shown  that  this  drug  is  the  most  de- 
structive agent  we  possess  as  an  amebicide.  Unfortunately  it  does 
not  kill  the  encysted  forms. 

Exposure  to  1:1000  solution  of  formalin  did  not  kill  encysted 
amebas  in  twenty-four  hours.  Acetozone  did  not  kill  amebas  in 
1:1000  dilutions.  If,  however,  the  acetozone  was  made  i  per  cent, 
acid  to  phenolphthalein  the  amebas  were  all  killed  by  i :  5000 
solutions  in  ten  minutes. 

Metabolic  Products. — -It  seems  as  though  Entamceba  histolytica 
must  produce  some  metabolic  product  that  exerts  an  enzymic  ac- 
tion upon  the  human  tissues  and  thus  accounts  for  the  destructive 
nature  of  the  lesions.  This  has  not,  however,  been  demonstrated 
as  yet. 

G.  Fully  developed  cyst  of  Entamoeba  tetragena  containing  four  nuclei  and 

one  mass  of  chromatin. 
H.  Illustrating  the  type  of  nucleus  as  observed  in  Entamceba  tetragena  in 

specimens  stained  with  Giemsa  stain. 
Lower  Group. — Amoeba  lobospinosa  stained  with  Delafield's  hematoxylin  after 

fixation  with  sublimate  alcohol, 
i,  2,  and  3.  Vegetative  organisms  showing  the  large  contractile  vacuole  and 

the  typical  nucleus  containing  a  deeply    stained    karyosome    almost 

filling  the  nucleus. 

4.  A  vegetative  ameba  in  whicl^  the  nucleus  has  divided. 

5,  6.  Vegetative  amebae  in  which  the  nucleus  is  dividing.     Polar  bodies 

are  present    connected  by  filaments  and  a  well-marked  equatorial 
plate  is  apparent. 

7.  Degenerated  vegetative  ameba  filled  with  vacuoles  and  with  atypically 

staining  nucleus. 

8.  Amoeba    lobospinosa    containing  a   protozoan  organism.     These   forms 

have  been  mistaken  for  sporulating  amebae. 

9  and  10.  Encysted  forms  of  Amoeba  lobospinosa  during  the  first  few 
days  in  cultures. 

ii  to  io  (except  14).  Various  cystic  forms  of  Amoeba  lobospinosa  show- 
ing the  character  of  the  cyst  wall  in  the  older  cysts.  At  12  the  cyst 
contains  two  vacuoles  and  the  cyst  membrane  is  folded  in,  an  appear- 
ance frequently  observed  in  cultures  which  have  become  dry;  15  and 
17  represent  cysts  in  which  the  cyst  wall  is  cracked  and  a  nucleus  can- 
not be  distinguished;  16  represents  a  cyst  filled  with  deeply  staining 
granules  of  chromatin  derived  from  the  degenerated  nucleus;  18  is  a 
cyst  in  which  only  the  cystic  membrane  is  visible,  the  ameba  having 
escaped  from  the  cyst. 

14.  A  fragmenting  ameba  frequently  mistaken  for  a  budding  organism  before 
the  separation  of  the  fragments  (Charles  F.  Craig,  M.  D.,  in  Journal 
of  Medical  Research,  vol.  xxvi,  No.  i,  April,  1912). 


Amebic  Dysentery 


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Methods  of 
eproduction 


* 


644  Dysentery 

Pathogenesis.— Schaudinn  was  the  first  to  prove  the  pathogenic 
action  of  the  organism.  He  inspissated  the  evacuations  of  a  case 
suffering  from  dysentery,  so  that  it  contained  considerable  numbers 
of  encysted  amebas.  When  this  was  fed  to  kittens  they  died  in  two 
weeks  with  the  typical  lesions  of  dysentery.  Musgrave  and  Clegg 
had  less  satisfactory  results  with  cats,  dogs,  and  other  laboratory 
animals,  but  were  quite  satisfied  with  the  results  secured  with 
monkeys,  which  took  the  disease  and  sometimes  died.  The  lesions 
resembled,  but  were  less  severe  than  those  in  man.  Musgrave  and 
Clegg  would  not  admit  that  there  were  non-pathogenic  intestinal 
amebas,  but  this  was  not  in  accord  with  the  work  of  any  other 
investigators,  and  was  strongly  opposed  by  Craig,*  who  found  both 

Secondary  abscesses 

Falciform  ligament 


Small  abscesses  

^ft^n^feL  ^^^^^^^~'    ^vy^K'x 

^  Secondary  abscess  in 

Main  abscess  Lymphatic  spigelian  lobe 

gland 

Fig.  262. — Multiple  amebic  abscesses\of  the  liver  (J.  E.  Thompson,  in  Interna- 
tional Clinics,  vol.  n,  i4th  Series,  J.  B.  Lippincott  Co.,  Publishers). 

varieties,  and  though  he  was  never  able  to  infect  animals  with 
Entamceba  coli,  was  successful  with  the  pathogenic  varieties,  and 
succeeded  in  infecting  50  per  cent,  of  the  kittens  he  experimented 
upon,  by  injecting  the  amebas  into  the  rectum. 

Lesions. — The  gross  morbid  appearances  of  the  intestinal  lesions 
in  both  forms  of  dysentery  are  sufficiently  distinct  in  typical  cases 
to  enable  an  experienced  pathologist  to  differentiate  them,  yet  not 
sufficiently  distinct  to  make  them  easy  of  description.  The  one 
great  characteristic  feature  of  the  amebic  dysentery  is  abscess  of 
the  liver  which  occurs  in  nearly  25  per  cent,  of  the  cases,  but  which 
almost  never  occurs  in  bacillary  dysentery. 

The  distinct  and  somewhat  rigid  ectoplasm  of  the  Entamceba 
histolytica  is  supposed  to  make  it  easy  for  the  organisms,  which  it 
*  "Journal  of  Infectious  Diseases,"  1908,  v,  p.  324. 


Amebic  Dysentery 


645 


I 


Figs.  263,  264. — Colon.     Tropical  or  amebic  dysentery. 


646 


Dysentery 


will  be  remembered  are  actively  motile,  to  penetrate  between  the 
epithelial  cells  of  the  intestinal  mucosa  to  the  lymph-spaces  of  the 
submucosa  below.  Here  the  amebas  multiply  in  large  numbers, 
and  by  the  enzymic  action  of  their  metabolic  products  produce 
necrosis  of  the  suprajacent  tissues  with  resulting  exfoliation  and 
the  production  of  round,  oval,  or  ragged  ulcerations  with  markedly 
infiltrated  and  undermined  edges.  As  the  amebas  continue  to 
increase  and  fill  up  the  lymphatics,  and  as  bacteria  add  their  effects 
to  those  occasioned  by  the  amebas,  the  ulcers  increase  in  extent 
and  depth  until  the  mucosa  and  submucosa  may  be  almost  entirely 


— *  Gf 


A  "' 


Fig.  265. — Entamoeba  histolytica.  Section  of  the  human  intestinal  wall 
showing  the  amebas  at  the  base  of  a  dysenteric  ulcer:  A,  A,  A,  Amebas,  some  of 
which  are  in  blood-vessels,  Gf  (Harris). 

destroyed,  leaving  the  entire  large  intestine  denuded,  except  for 
occasional  islands  of  much  congested,  inflamed,  and  partly  necrotic 
mucous  membrane!  The  diseased  intestinal  wall  is  the  seat  of  much 
congestion  and  is  much  thickened.  The  amebas  not  only  occur  in 
great  numbers  in  the  interstices  of  the  tissues  about  the  base  of  the 
ulcers  and  in  the  lymphatics,  but  also  enter  the  capillaries,  through 
which  they  are  carried  to  the  larger  vessels,  and  eventually  to  the 
liver,  where  their  activities  continue  and  give  rise  to  the  amebic 
abscess.  The  first  expression  of  their  injury  to  the  liver  parenchyma 
is  shown  by  focal  necroses.  In  each  of  these  the  organisms  multiply 
and  the  lesion  extends  until  neighboring  necroses  are  brought 
into  union,  and  eventuate  in  great  collections  of  colliquated  necrotic 


Bacillary  Dysentery  647 

material  which  may  be  so  extensive  as  to  involve  the  entire  thick- 
ness of  the  organ.  There  is  usually  one  large  abscess,  but  there 
may  be  several  small  ones,  or  the  liver  may  be  riddled  with  minute 
abscesses.  The  content  of  the  abscesses  is  pinkish  necrotic  material 
in  which  amebas  are  few.  The  walls  are  of  semi-necrotic  material, 
in  which  great  numbers  of  amebas  abound.  The  liver  sometimes 
becomes  adherent  to  the  diaphragm,  may  perforate  it,  and  after 
adhesion  of  the  lung  to  the  diaphragm  may  evacuate  through  the 
lung,  the  pinkish  abscess  contents  with  amebas  being  expectorated. 
Sections  of  the  intestinal  wall  and  of  the  liver  near  the  border 
of  the  abscess  show  the  amebas  well  when  stained  with  iron-hema- 
toxylon,  or  perhaps  still  better  by  Mallory's  differential  method.* 

1.  Harden  the  tissue  in  alcohol. 

2.  Stain   sections   in    a  saturated  aqueous  solution  of  thionin  three  to  five 
minutes. 

3.  Differentiate  in  a  2  per  cent,  aqueous  solution  of  oxalic  acid  for  one-half  to 
one  minute. 

4.  Wash  in  water. 

5.  Dehydrate  in  absolute  alcohol. 

6.  Clear  in  alcohol. 

7.  Xylol-balsam. 

The  nuclei  of  the  amebas  and  the  granules  of  the  mast-cells  are  stained  brown- 
ish red;  the  nuclei  of  the  mast-cells  and  of  all  other  cells  are  stained  blue. 


II.  BACILLARY  DYSENTERY 

BACILLUS  DYSENTERIC  (SHIGA) 

General  Characteristics. — A  non-motile,  non-flagellated,  non-sporogenous, 
non-liquefying,  aerobic  and  optionally  anaerobic,  non-chromogenic,  non-aero- 
genic,  pathogenic  bacillus  of  the  intestine,  staining  by  ordinary  methods,  but  not 
by  Gram's  method.  It  does  not  produce  indol.  It  first  acidifies,  then  alkalin- 
izes  milk,  but  does  not  coagulate  it. 

After  considerable  investigation  of  the  epidemic  dysentery 
prevalent  in  Japan,  Shigaf  came  to  the  conclusion  that  a  bacillus 
which  he  called  Bacillus  dysenteriae  was  its  specific  cause. 

It  is  not  improbable  that  the  bacillus  of  Shiga  is  identical  with 
Bacterium  coli,  variety  dy sentence,  of  Celli,  Fiocca,  and  Scala,J 
a  view  that  has  been  further  confirmed  by  Flexner.||  It  may  also 
be  identical  with  an  organism  described  in  1888  by  Chantemasse 
and  Widal.§ 

In  1899  Flexner,**  while  visiting  the  Philippine  Islands,  isolated 
a  bacillus  from  the  epidemic  dysentery  prevailing  there,  which  he 
regarded  as  identical  with  Shiga's  organism.  In  1890  Strong  and 

*  "Pathological  Technic,"  1911,  p.  434. 

t  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1898,  xxrv,  Nos.  22-24. 
j  "Hygien.  Institut.  Rom.  Univ.,"  1895,  and  "Centralbl.  f.  Bakt.  u.  Parasi- 
tenk.," 1899. 

"Univ.  of  Penna.  Med.  Bulletin,"  Aug.,  1901. 
Deutsche  med.  Wochenschrift,"  1903,  No.  12. 
"Bulletin  of  the  Johns  Hopkins  Hospital,"  1900,  rx. 


648  Dysentery 

Musgrave*  isolated  what  appeared  to  be  the  same  organism,  also 
from  cases  of  dysentery  in  the  Philippines.  Almost  at  the  same 
time  Krusef  was  investigating  an  epidemic  of  dysentery  in  Germany, 
and  succeeded  in  isolating  a  bacillus  that  also  bore  fair  correspond- 
ence to  that  of  Shiga.  In  1901  SpronckJ  found  a  bacillus  in 
cases  of  dysentery  occurring  in  Utrecht,  Holland,  that  corresponded 
with  a  slightly  different  organism  first  found  and  described  by  Kruse§ 
•as  a  "pseudodysentery  bacillus." 

In  1902  Park  and  Dunham ||  investigated  a  small  epidemic  of 
dysentery  in  Maine,  and  there  found  a  bacillus  similar  to  those  al- 
ready described.  In  1903  Hiss  and  Russell  described  a  bacillus 
"  Y"  from  a  case  of  fatal  diarrhea  in  a  child. 

Bacillus  dysenteriae  was  also  found  by  Vedder  and  Duval**  in 
the  epidemic  and  sporadic  dysentery  of  the  United  States.  Duval 
and  Bassetttf  and  Martha  WollsteinJt  found  Bacillus  dysenteriae  in 
cases  of  the  summer  diarrheas  of  infants,  especially  when  such  diar- 
rheas were  epidemic. 

Lentz§§  has  shown  that  dysentery  and  pseudodysentery  bacilli 
present  differences  in  their  behavior  toward  sugars.  Various  ob- 
servers found  differences  in  the  behavior  of  the  various  bacilli  to 
the  agglutinating  effects  of  artificially  prepared  immune  serum. 
The  outcome  of  these  investigations  is  the  discovery  that  Bacillus 
dysenteriae  is  a  species  in  which  there  are  a  number  of  different 
varieties  well  characterized,  but  by  differences  too  slight  to  permit 
them  to  be  regarded  as  separate  species.  This  thought — that  we 
are  dealing  with  a  group  of  varieties  and  not  a  single  well-defined 
organism — is  essential  to  an  intelligent  understanding  of  the  bacteri- 
ology of  dysentery. 

Varieties  of  the  Dysentery  Bacillus. — Three  varieties  of  the 
dysentery  bacillus  may  now  be  described: 

1.  The  Shiga- Kruse  variety. 

2.  The  Flexner  variety. 

3.  The  Hiss-Russell  variety. 

The  differences  by  which  they  are  separated  are  to  be  found 
in  their  varying  agglutinability  by  artificially  prepared  immune 
serums,  each  of  which  exerts  a  far  more  pronounced  effect  upon  its 
own  variety  than  upon  the  others,  and  in  the  behavior  toward  sugars 
with  reference  to  acid  formation  and  gas  production.  It  seems  not 
improbable  that  the  future  will  have  much  to  say  about  the  dys- 

*  "Report  Surg.  Gen.  U.  S.  Army,"  Washington,  1900. 

t  "Deutsche  med.  Wochenschrift,"  1900,  xxvi. 

j"Ref.  Baumgarten's  Jahresberichte,"  1901.     . 

§  "Deutsche  med.  Wochenschrift,"  1901,  Nos.  23  and  24. 

I)  "New  York  Bull,  of  Med.  Sciences,"  1902. 

**  "Journal  of  Experimental  Medicine,"  1902;  vol.  vi,  No.  2,  "American  Medi- 
cine," 1902. 

tf  "American  Medicine,"  Sept.  13,  1902,  vol.  iv,  No.  11,  p.  417. 
It  "Jour.  Med.  Research,"  1904,  x,  p.  n. 
§§  "Zeitschrift  f.  Hygiene,"  etc.,  1902,  XLI. 


Bacillary  Dysentery  649 

entery  bacillus,  and  that  the  validity  of  much  that  is  accepted  at 
present  may  have  to  be  amended.  This  seems  to  be  particularly 
true  with  regard  to  the  matter  of  fermentation,  the  details  of  which 
are  displayed  in  the  table  taken  from  Muir  and  Ritchie's  "  Manual 
of  Bacteriology"  (p.  650). 

Morphology. — -The  organism  is  a  short  rod  with  rounded  ends, 
generally  similar  to  the  typhoid  bacilli.  It  usually  occurs  singly, 
but  may  occur  in  pairs.  It  is  frequently  subject  to  involutional 
changes.  It  is  doubtfully  motile  and  is  probably  without  flagella. 

Staining. — When  stained  with  methylene-blue  the  ends  color 
more  deeply  than  the  middle;  and  organisms  from  old  cultures 
show  numerous  involution  forms  and  irregularities.  It  stains 
with  ordinary  solutions,  but  not  by  Gram's  method.  It  has  no 
spores. 

Cultivation. — The  organism  grows  well  in  slightly  alkaline  media 
under  aerobic  conditions. 

Colonies. — The  colonies  upon  gelatin  plates  are  small  and  dew- 
drop-like  in  appearance.  Upon  microscopic  examination  they  are 
seen  to  be  regular  and  of  spheric  form.  By  transmitted  light  they 
appear  granular  and  of  a  yellowish  color.  They  do  not  spread  out 
in  a  thin  pellicle  like  those  of  the  colon  bacillus,  and  there  are  no 
essential  differences  between  superficial  and  deep  colonies. 

Gelatin  Punctures. — The  growth  in  the  puncture  culture  consists 
of  crowded,  rounded  colonies  along  the  puncture.  A  grayish-white 
growth  forms  upon  the  surface.  There  is  no  liquefaction  of  the 
medium. 

Agar-agar. — Upon  the  surface  of  agar-agar,  cultures  kept  in 
the  incubating  oven  show  large  solitary  colonies  at  the  end  of 
twenty-four  hours.  They  are  bluish-white  in  color  and  rounded 
in  form.  The  surface  appears  moist.  In  the  course  of  forty- 
eight  hours  a  transparent  border  is  observed  about  each  colony,  and 
the  bacilli  of  which  it  is  composed  cease  to  stain  evenly,  presenting 
involution  forms. 

Glycerin  agar-agar  seems  less  well  adapted  to  their  growth  than 
plain  agar-agar.  Blood-serum  is  not  a  suitable  medium. 

Litmus  Milk. — Milk  is  not  coagulated.  As  the  growth  progresses 
there  is  slight  primary  acidity,  which  later  gives  place  to  an  in- 
creasing alkalinity. 

Potato. — -Upon  boiled  potato  the  young  growth  resembles  that 
of  the  typhoid  bacillus,  but  after  twenty-four  hours  it  becomes 
yellowish  brown,  and  at  the  end  of  a  week  forms  a  thick,  brownish- 
pink  pellicle. 

Bouillon. — In  bouillon  the  bacillus  grows  well,  clouding  the 
liquid.  No  pellicle  forms  on  the  surface. 

Metabolic  Products. — The  organism  does  not  form  indol,  does 
not  ferment  dextrose,  lactose,  saccharose,  or  other  carbohydrates. 


650 


Dysentery 


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+  in  Motility  column 
—  in  Motility  columi 
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"A"  =  acid  product 
*  McConkey,"Journ 

Bacillary  Dysentery  651 

Acids  are  produced  in  moderate  quantities  after  twenty-four  hours. 
Milk  is  not  coagulated.  Gelatin  is  not  liquefied. 

Toxins,  chiefly  endotoxins,  are  produced.  They  may  best  be 
prepared  by  making  massive  agar-agar  cultures  in  Kitasato  flasks 
or  flat-sided  bottles,  and  after  growth  is  complete  washing  off  the 
bacillary  mass  with  a  very  small  quantity  of  sterile  salt  solu- 
tion, and  after  killing  the  bacilli  by  exposure  to  6o°C.  for  fifteen  to 
thirty  minutes,  permitting  the  rich  suspension  to  autolyze  for 
three  days.  The  toxins  may  be  precipitated  from  the  sodium 
chlorid  solution  by  ammonium  sulphate. 

Vital  Resistance.— The  thermal  death-point  is  68°C.  maintained 
for  twenty  minutes.  It  grows  slowly  at  ordinary  temperatures, 
rapidly  at  the  temperature  of  the  body. 

Pathogenesis. — 'Shiga  and  Flexner  found  that  infection  of  young 
cats  and  dogs  could  be  effected  by  bacilli  introduced  into  the  stom- 
ach, and  that  lesions  suggestive  of  human  dysentery  were  present  in 
the  intestines.  Kazarinow*  found  that  when  guinea-pigs  and 
young  rabbits  were  narcotized  with  opium,  the  gastric  contents 
alkalinized  with  10  cc.  of  a  10  per  cent.  NaOH  solution,  and  a 
quantity  of  Shiga  bacilli  introduced  into  the  stomach  with  an 
esophageal  bougie,  it  was  possible  to  bring  about  diarrhea  and 
death  with  lesions  similar  to  those  described  by  Vaillard  and  Dopter. 

In  these  experiments  it  was  found  that  rapid  passage  through 
animals  greatly  increased  the  virulence  of  the  bacilli,  and  it  was 
also  observed  that  though  0.0005  cc-  °f  a  virulent  culture  intro- 
duced into  the  peritoneal  cavity  would  cause  fatal  infection,  to 
produce  infection  by  the  mouth  as  above  stated  required  the  en- 
tire mass  of  organisms  grown  in  five  whole  culture-tubes. 

The  virulent  organisms  are  infectious  for  guinea-pigs  and  other 
laboratory  animals,  and  cause  fatal  generalized  infection  without 
intestinal  lesions. 

Lesions. — The  lesions  found  in  human  dysentery  are  usually 
fairly  destructive.  They  consist  of  a  severe  catarrhal  and  pseudo- 
membranous  colitis,  which  later  passes  into  a  stage  of  marked 
ulceration.  There  is  great  thickening  of  the  submucosa  and  the 
whole  of  the  intestinal  lining  is  corrugated.  For  the  most  part  the 
ulcerations  are  more  superficial  than  those  of  the  amebic  dysentery, 
and  the  edges  of  the  ulcerations  show  less  tumefaction  and  less 
undermining.  Abscess  of  the  liver  does  not  occur  in  bacillary 
dysentery. 

Diagnosis. — The  blood-serum  of  those  suffering  from  epidemic 
dysentery  or  from  those  recently  recovered  from  it  causes  a  well- 
marked  agglutinative  reaction.  This  agglutination  was  first  care- 
fully studied  by  Flexner,  and  is  peculiar  in  that  the  serums  pre- 
pared from  the  different  varieties  of  the  bacillus,  while  they  exert 

*  "Archiv.  f.  Hyg.,"  Bd.  L,  Heft  i,  p.  66;  see  also  "Bull,  de  1'Inst.  Past.,"  15 
Aout,  1904,  p.  634. 


652  Balantidium  Coli 

some  action  upon  all  varieties  of  the  organism,  exert  a  much  more 
powerful  influence  upon  the  particular  variety  used  in  their  prepa- 
ration. The  same  is  true  of  the  patient's  serum,  hence,  in  making 
use  of  the  agglutination  reaction  for  the  diagnosis  of  the  disease, 
the  blood  of  the  patient  should  be  tested  by  contact  with  all  of 
the  different  cultures. 

Serum  Therapy.— By  the  progressive  immunization  of  horses 
to  an  immunizing  fluid,  the  basis  of  which  is  a  twenty-four-hour- 
old  agar-agar  culture  dried  in  vacua,  Shiga  prepared  an  antitoxic 
serum  with  which,  in  1898,  in  the  Laboratory  Hospital  65  cases 
were  treated,  with  a  death-rate  of  9  per  cent.;  in  1899,  in  the  Labo- 
ratory Hospital,  91  cases,  with  a  death-rate  of  8  per  cent.;  in  1899, 
in  the  Hirowo  Hospital,  no  cases,  with  a  death-rate  of  12  per 
cent.  These  results  are  very  significant,  as  the  death-rate  in 
2736  cases  simultaneously  treated  without  the  serum  averaged 
34.7  per  cent.,  and  in  consideration  of  the  frequency  and  high  death- 
rate  of  the  disease,  Japan  alone,  between  the  years  1878  and  1899, 
furnishing  a  total  of  1,136,096  cases,  with  275,308  deaths  (a  total 
mortality  for  the  entire  period  of  24.23  per  cent.).* 

BALANTZDIUM  DIARRHEA 

BALANTIDIUM  COLI  (MALMSTEN) 

In  certain  rare  cases  a  severe  form  of  diarrhea,  or  a  mild  form  of  dysentery 
appears  to  depend  neither  upon  Entamoeba  histolytica  nor  Bacillus  dysenteriae, 
but  upon  an  infusorian  parasite  known  as  Balantidium  coli.  This  organism  was 
first  observed  by  Malmstenf  in  1857  in  the  intestines  of  a  man  who  had  suffered 
from  cholera  two  years  before  and  had  ever  since  suffered  from  diarrhea.  Upon 
investigation,  an  ulceration  was  found  in  the  rectum  just  above  the  internal 
sphincter.  In  the  bloody  pus  from  this  ulcer  numerous  balantidia  were  seen 
swimming  about.  Although  the  ulcer  healed,  the  diarrhea  did  not  cease.  Since 
this  original  observation  and  up  to  igo8,Brauni  had  beenableto  collect  142  cases 
of  human  infection.  In  all  of  theseN  cases  the  presence  of  the  balantidium  was 
accompanied  by  obstinate  diarrhea  with  bloody  discharges  (dysentery)  in  some, 
and  many  of  the  cases  ended  in  death. 

Morphology. — 'The  Balantidium  coli  is  a  ciliate  protozoan  micro-organism  of 
ovoid  or  ellipsoidal  form,  measuring  from  30  to  200  p  in  length  and  from  20  to 
70  ju  in  breadth.  The  body  is  surrounded  by  a  distinct  ectosarc  completely 
covered  by  short  fine  cilia.  The  anterior  end,  which  is  usually  a  little  sharper 
than  the  posterior,  presents  a  deep  indentation,  the  peristome,  which  continues, 
in  an  infundibuliform  manner,  deeply  into  the  endosarc.  The  peristome  is 
surrounded  by  a  circle  of  longer  cilia — adoral  cilia — than  those  elsewhere  upon 
the  body.  At  the  opposite  pole  there  is  a  small  opening  in  the  ectosarc,  the 
anus.  The  mouth  is  the  simple  termination  of  the  infundibuliform  extension  of 
the  peristome  and  opens  directly  into  the  endosarc,  so  that  the  small  bodies  upon 
which  the  organism  feeds,  and  which  are  continually  being  caught  in  the  vortex 
caused  by  the  rapidly  vibrating  adoral  cilia  are  driven  down  the  short  tubulature 
directly  into  the  endosarc. 

The  endosarc  is  granular  and  contains  fat  and  mucin  granules,  starch  grains, 
bacteria,  and  occasionally  red  and  white  blood-corpuscles. 

There  are  usually  two  contractile  vacuoles,  sometimes  more,  and  as  the  quiet 

*  "Public  Health  Reports,"  Jan.  5,  1900,  vol.  xv,  No.  i. 

f  "  Archiv.  f.  pathologische  Anatomie,"  etc.,  xn,  1857,  p.  302. 

j  "Tierische  Parasiten  des  Menschen,"  Wurzburg,  1908. 


Balantidium  Diarrhea 


653 


organism  is  watched  these  large  clear  spaces  can  be  seen  alternately  to  contract 
and  expand. 

There  are  two  nuclei.  The  larger,  or  macronucleus,  is  bean-shaped,  kidney- 
shaped,  or,  more  rarely,  oval.  The  smaller,  the  micronucleus,  is  spherical. 
There  is  no  digestive  tube;  the  nutritious  particles  are  directly  in  the  endosarc,  in 
which  they  are  digested,  any  residuum  being  extruded  from  the  anus. 

Motility. — The  organism  is  actively  motile,  swimming  rapidly  at  a  steady  pace 
or  darting  here  and  there. 

Staining. — The  organism  can  be  most  easily  and  satisfactorily  studied  while 
alive.  To  stain  it  a  drop  of  the  fluid  containing  the  balantidia  is  spread  upon  a 
slide  and  permitted  to  dry.  Just  before  the  moisture  disappears  from  the  film, 
methyl  alcohol  may  be  poured  upon  it  to  kill  and  fix  the  organisms.  The  staining 
may  then  be  performed  with  Giemsa's  polychrome  methylene-blue  or  iron-hema- 
toxylon.  The  cilia  usually  do  not  show. 


Fig.  266. — Reproduction  of  Balantidium  coli:  1-5,  Asexual  reproduction  by 
division;  6,  encysted  form  of  single  individuals;  7,  conjugation  of  two  individu- 
als; 8,  reproductive  cyst;  9,  cyst  with  peculiar  contents  whose  further  develop- 
ment has  not  been  followed  (Brumpt). 

Reproduction. — This  commonly  takes  place  by  karyokinesis,  followed  by  trans- 
verse division,  and  in  cases  of  experimental  infection  so  rapidly  that  the  organ- 
isms have  not  time  to  grow  to  the  full  size  before  dividing  again.  The  result  is 
that  many  appear  that  are  no  more  than  30  ju.  in  length.  In  addition  to  multi- 
plication by  division,  there  is  a  sexual  cycle  of  development  with  conjugation. 
This  was  first  pointed  out  by  Gourvitsch,*  studied  by  Leger  and  Duboscq,f  and 
further  confirmed  by  Brumpt.  %  In  the  process  of  conjugation  two  individuals 
come  together,  become  attached  lengthwise,  and  fuse  into  a  single  large  organism 
that  forms  a  cyst  several  times  as  large  as  a  balantidium,  and  with  contents  no 
longer  recognizable  as  such.  The  contents  of  this  cyst  eventually  divide  into  a 
number  of  spheres,  but  how  these  subsequently  develop  appears  not  to  have  been 
determined. 

*  "Russ.  Archiv  f.  Path.  klin.  Med.  u.  Bact.  St.  Petersb.,"  1896,  quoted  by 
Braun. 

t  "Archiv  de  Zool.  Exper.,"  1904,  n,  No.  4. 

j  "  Compt.-rendu  de  la  Soc.  de  Biol.,"  July  10,  1909. 


654 


Balantidium  Coli 


Habitat. — The  balantidium  is  unknown  except  as  a  parasite  of  the  colon.  It  is 
very  common  in  hogs  and  has  been  found  in  the  orang-outang,  in  certain  lower 
monkeys  (Macacus  cynomolgus),  and  in  man. 

Cultivation. — The  organism  quickly  dies  when  transplanted  to  artificial  media 
and  has  not  yet  been  cultivated  artificially. 

Pathpgenesis. — The  presence  of  the  organisms,  in  whatever  kind  of  animal, 
gives  rise  to  colitis,  which  is  at  first  catarrhal,  but  soon  becomes  more  or  less 
ulcerative.  Some  doubt  has  been  expressed  as  to  the  exact  role  of  the  balantidia 
in  the  causation  of  the  inflammation,  some  believing  them  to  be  rather  acci- 
dental factors  than  the  true  etiologic  excitants.  As  the  organisms  descend  into 
the  ulcerated  tissues  and  from  the  denuded  surfaces  invade  the  lymphatics, 
there  seems  to  be  little  doubt  of  their  pathogenic  importance. 

Animal  Inoculation. — Experiments  made  by  Casagrandi  and  Barbagallo,* 
Klimenko,t  and  others  upon  kittens  and  pups  have  failed  to  produce  the  disease 


Fig.  267. — Balantidium  coli  deeply  situated  in  the  interglandular  tissue  of  the 
intestinal  mucosa  (Brumpt). 


even  when  the  colon  was  already  inflamed.  Brumpt,  J  on  the  contrary,  suc- 
ceeded in  reproducing  it  in  monkeys  and  pigs  by  introducing  the  encysted 
organisms  into  the  already  inflamed  intestine  via  the  anus. 

Lesions. — In  the  majority  of  fatal  cases  postmortem  examination  of  the  colon 
shows  it  to  be  in  a  state  of  catarrhal  inflammation  with  numerous  superficial 
ulcerations  with  considerable  surrounding  infiltration  of  the  mucosa.  Twenty- 
four  hours  from  the  time  of  the  death  of  the  patient  the  balantidia  are  all  dead. 
Strong  and  Musgrave,||  Solowiew,§  Klimenko,**  and  others  have  shown  that  in 
microscopic  sections  of  the  inflamed  tissues  the  micro-organisms  could  be  found 
deep  down  in  the  blood-vessels  and  lymphatic  spaces  about  the  ulcerated  areas, 
sometimes  penetrating  as  deeply  as  the  serous  coat  of  the  bowel.  Metastatic 


'Bal.  coli,"  etc.,  Catania,  1896,  quoted  by  Braun. 
'Beitrage  zur.  path.  Anat.  u.  allg.  Path.,"  1903,  xxxn,  281. 
'Precis  de  Parasitology,"  1910,  152. 
'Bulletin  of  the  Johns  Hopkins  Hospital,"  1901,  xn,  31. 
'Centralbl.  f.  Bakt.,"  etc.,  i  Abl.,  1901,  xxrx,  821,  849. 
Loc.  cit. 


Balantidium  Diarrhea  655 

abscess  of  the  liver  may  be  caused  by  balantidia,  and  has  been  reported  by 
Manson,*  and  a  case  of  abscess  of  the  lung  caused  by  the  organism  by  Wino- 
gradow  and  Stokvis.f 

Transmission. — The  transmission  of  the  disease  can  only  come  about  through 
the  encysted  form  of  the  parasites.  Great  numbers  are  passed  in  the  feces  of  the 
infected  animals,  but  except  the  encysted  forms  all  die  very  quickly  as  the  fecal 
matter  dries.  Unfortunately  the  further  life-history  of  the  encysted  forms  is 
unknown. 

CRAIGIA  HOMINIS  (Calkins^ 

Craigia  hominis  is  an  ameboid  and  flagellated  intestinal  protozoan  parasite  of 
man,  described  in  1906  by  Craig§  and  recently  carefully  and  elaborately  studied 
by  Barlow.  ||  It  is  a  minute  organism  and  has  an  amebic  stage  during  which  it 
reproduces  by  simple  division  like  a  typical  ameba  for  several  generations  or 
as  long  as  conditions  are  favorable.  It  then  encysts,  and  within  the  cysts 
numerous  small  bodies  called  "swarmers"  develop  and  escape.  Each  of  these 
has  a  long  single  protoplasmic  flagellum  and  is  actively  motile.  The  swarmers 


Fig.  268.—  Craigia  hominis  (Barlow,  in  American  Journal  of  Tropical  Diseases). 


multiply  by  longitudinal  division  for  several  generations  after  which  the  flagella 
disappear  and  the  amebic  stage  begins  again. 

In  56  cases  of  infection  by  this  parasite  studied  by  Barlow,  diarrhea  was  the 
most  invariable  symptom.  Enterrhagia  is  less  frequent  and  less  severe  in 
craigiosis  than  in  amebiasis.  Of  the  56  cases,  n  developed  abscess  of  the  liver, 
one  a  pulmonary  abscess,  two  appendicitis,  one  arthritis,  two  duodenal  ulcer, 
while  others  had  more  vague  complications  and  sequelae.  It  seems,  from  Barlow's 
studies,  that  the  parasite  deserves  considerable  attention.  The  discovery  of  the 
parasite  was  made  in  the  Philippine  Islands,  but  Barlow's  cases  were  in  Hon- 
duras. One  case  has  been  reported  in  Texas,  another  in  Tennessee. 

Barlow  recognizes  two  species,  Craigia  hominis  and  Craigia  migrans. 

HARMLESS  FLAGELLATES  OF  THE  HUMAN  INTESTINES 

In  certain  cases  of  diarrhea,  flagellates  —  Trichomonas  intestinalis,  Cercomonas 
intestinalis,  and  Lamblia  (Megastomum)  intestinalis  have  been  discovered.  As, 
however,  they  seem  to  be  frequent  denizens  of  normal  intestines,  it  is  doubtful 
whether  their  presence  is  more  than  incidental. 

*  "Tropical  Diseases,"  1900,  p.  394. 

t  "Niederl.  Tijdschr.  v.  Geneeskde.,"  1884,  xx,  No.  2,  quoted  by  Braun. 

j  Trans,  xvth  Internal.  Congress  of  Hygiene  and  Demography,  1912,  n,  287. 

§Amer.  Jour.  Med.  Sciences,  1906,  cxxxn,  214. 

||  The  American  Journal  of  Tropical  Diseases,  etc.,  1915,  n,  680. 


CHAPTER  XXIX 
TUBERCULOSIS 

BACILLUS  TUBERCULOSIS  (KOCH) 

General  Characteristics. — A  non-motile,  non-flagellate,  non-sporogenous,  non- 
liquefying,    npn-chromogenic,    non-aerogenic,     distinctly    aerobic,    acid-proof, 
Sirely  parasitic,  highly  pathogenic  organism,  staining  by  special  methods  and  by 
ram's  method.     Commonly  occurring  in  the  form  of  slender,  slightly  curved 
rods  with  rounded  ends,  not  infrequently  showing  branches,  hence  probably  not  a 
bacillus,  but  an  organism  belonging  to  the  higher  bacteria.     It  does  not  produce 
indol  or  acidulate  or  coagulate  milk. 

Tuberculosis  is  one  of  the  most  destructive  and,  unfortunately, 
one  of  the  most  common  diseases.  It  is  no  respecter  of  persons, 
but  affects  alike  the  young  and  old,  the  rich  and  poor,  the  male  and 
female,  the  enlightened  and  savage,  the  human  being  and  the 
lower  animals.  It  is  the  most  common  cause  of  death  among  human 
beings,  and  is  common  among  animals,  occurring  with  great  fre- 
quency among  cattle,  less  frequently  among  goats  and  hogs,  and 
sometimes,  though  rarely,  among  sheep,  horses,  dogs,  and  cats. 

Wild  animals  under  natural  conditions  seem  to  escape  the  dis- 
ease, but  when  caged  and  kept  in  zoologic  gardens,  even  the  most 
resistant  of  them — lions,  tigers,  etc. — are  said  at  times  to  succumb 
to  it,  while  it  is  the  most  common  cause  of  death  among  captive 
monkeys. 

The  disease  is  not  limited  to  mammals,  but  occurs  in  a  some- 
what modified  form  in  birds,  and  it  is  said  even  at  times  to  affect 
reptiles,  batrachians  and  fishes. 

The  disease  has  been  recognized  for  centuries;  and  though, 
before  the  advent  of  the  micrescope,  it  was  not  always  clearly 
differentiated  from  cancer,  it  has  not  only  left  unmistakable  signs 
of  its  existence  in  the  early  literature  of  medicine,  but  has  also  im- 
printed itself  upon  the  statute-books  of  some  countries,  as  the 
kingdom  of  Naples,  where  its  ravages  were  great  and  the  means 
taken  for  its  prevention  radical. 

Specific  Organism. — Although  the  acute  men  of  the  early  days 
of  pathology  clearly  saw  that  the  time  must  come  when  the  parasitic 
nature  of  tuberculosis  would  be  proved,  and  Klebs,  Villemin,  and 
Cohnheim  were  "within  an  ace"  of  its  discovery,  and  Baumgarten* 
probably  saw  it  in  tissues  cleared  with  lye,  it  remained  for  Robert 
Kochf  to  demonstrate  and  isolate  the  Bacillus  tuberculosis,  the 
specific  cause  of  the  disease,  and  to  write  so  accurate  a  description 

*  "Virchow's  Archives,"  Bd.  LXXXII,  p.  397. 
t  "Berliner  klin.  Wochenschrift,"  1882,  15. 
656 


Morphology  657 

of  the  organism,  and  the  lesions  it  produces,  as  to  be  almost  without 
a  parallel  in  medical  literature. 

Distribution. — So  far  as  is  known,  the  tubercle  bacillus  is  a 
purely  parasitic  organism.  It  has  never  been  found  except  in  the 
bodies  and  discharges  of  animals  affected  with  tuberculosis,  and 
in  dusts  of  which  these  are  component  parts.  This  purely  parasitic 
nature  interferes  with  the  isolation  of  the  organism,  which  cannot 
be  grown  upon  the  ordinary  culture-media. 

The  widespread  distribution  of  tuberculosis  at  one  time  sug- 
gested that  tubercle  bacilli  were  ubiquitous  in  the  atmosphere,  that 
we  all  inhaled  them,  and  that  it  was  only  our  vital  resistance  that 
prevented  us  all  from  becoming  its  victims.  Cornet,*  however, 


**  /^'Vr'i'l' 
i&i&M?. 


Fig.  269.— Tubercle  bacillus  in  sputum  (Frankel  and  Pfeiffer). 

showed  the  bacilli  to  be  present  only  in  dusts  with  which  pulverized 
sputum  was  mixed,  and  to  be  most  common  where  the  greatest 
uncleanliness  prevailed. 

Morphology. — The  tubercle  bacillus  is  a  slender,  rod-shaped 
organism  with  slightly  rounded  ends  and  a  slight  curve.  It  meas- 
ures from  1.5  to  3.5  IJL  in  length  and  from  0.2  to  o. 5  jj,  in  breadth. 
It  commonly  occurs  in  pairs,  which  may  be  associated  end  to  end, 
but  generally  overlap  somewhat  and  are  not  attached  to  each 
other.  Organisms  found  in  old  pus  and  sputum  show  a  peculiar 
beaded  appearance  caused  by  fragmentation  of  the  protoplasm  and 
the  presence  of  metachromatic  granules.  The  tubercle  bacillus 
forms  no  endospores. 

The  fragments,  originally  thought  by  Koch  to  be  spores,  are 
irregular  in  shape,  have  ragged  surfaces,  and  are  without  the  high 
refraction  peculiar  to  spores.  Spores  also  resist  heat  strongly,  but 

*  "Zeitschrift  fur  Hygiene,"  1888,  v,  pp.  191-331. 

42 


658  Tuberculosis 

the  fragmented  bacilli  are  no  more  capable  of  resisting  heat  than 
others. 

The  bacilli  not  infrequently  present  projecting  processes  or 
branches,  this  observation  having  changed  our  views  regarding  the 
classification  of  the  organism,  which  is  probably  erroneously  placed 
among  the  bacilli,  belonging  more  properly  to  the  higher  bacteria. 

The  organism  is  not  motile,  and  does  not  possess  flagella. 

Staining. — The  tubercle  bacillus  belongs  to  a  group  of  organisms 
which,  because  of  their  peculiar  behavior  toward  stains,  are  known 
as  "saurefest"  or  acid-proof.  It  is  difficult  to  stain  after  it  has 
lived  long  enough  to  invest  itself  with  a  waxy  capsule,  requiring  that 
the  dye  used  shall  contain  a  mordant  (Koch).  It  is  also  tenacious 
of  color  once  assumed,  resisting  the  decolorizing  power  of  strong 
mineral  acids  (Ehrlich). 


Fig.  270. — Bacillus  of  tuberculosis,N  showing  branched  forms  with  involution 

(Migula). 

Koch*  *first  stained  the  bacillus  with  a  solution  consisting  of 
i  cc.  of  a  concentrated  solution  of  methylene  bjue  mixed  with  20 
cc.  of  distilled  water,  well  shaken,  and  then,  before  using,  receiving 
an  addition  of  2  cc.  of  a  10  per  cent,  solution  of  caustic  potash. 
Cover-glasses  were  allowed  to  remain  in  this  for  twenty-four  hours 
and  subsequently  counterstained  with  vesuvin.  Ehrlich  subse- 
quently modified  Koch's  method,  showing  that  pure  anilin  was  a 
better  mordant  than  potassium  hydrate,  and  that  the  use  of  a 
strong  mineral  acid  would  remove  the  color  from  everything  but 
the  tubercle  bacillus.  This  modification  of  Koch's  method,  given 
us  by  Ehrlich,  probably  remains  the  best  method  of  staining  the 
bacillus. 

*  "  Mittheilungen  aus  dem  Kaiserlichen  Gesundheitsamte,"  1884,  n. 


Staining  659 

Nearly  all  of  the  recent  methods  of  staining  are  based  upon 
the  impenetrability  of  the  bacillary  substance  by  mineral  acids  which 
characterizes  the  acid-fast  or  acid-proof  (saurefest)  micro-organisms. 
But  it  is  not  improbable  that  we  have  been  led  into  error  by  the 
assumption,  upon  inadequate  grounds,  that  this  is  a  constant  and 
uniform  quality  of  the  tubercle  bacillus  and  similar  micro-organisms. 
The  interesting  observations  of  Much*  have  shown  that  many  of 
the  paradoxes  of  tuberculosis  can  be  accounted  for  by  the  fact  that 
during  certain  stages,  or  under  certain  conditions,  the  bacilli  are  not 
acid-proof  at  all.  Thus,  caseous  masses  from  the  lungs  of  cattle 
show  complete  absence  of  tubercle  bacilli  when  examined  by 
the  usual  method,  yet  cause  typical  tuberculosis  when  implanted 
into  guinea-pigs,  with  typical  bacilli,  recoverable  upon  culture- 
media,  in  the  lesions.  This  is  certainly  due  to  the  inability  of  the 
bacilli  in  the  bovine  lesions  mentioned  to  endure  the  acids,  for 
when  the  same  tissues  are  stained  by  Gram's  method  many  organ- 
isms can  be  found.  This  shows  that  Gram's  method  is  really  a 
more  useful  method  for  demonstrating  the  bacillus  than  those  in 
which  acids  are  employed.  Much  has  found  two  forms  of  the 
tubercle  bacillus,  one  rod-like,  the  other  granular,  that  are  not 
acid-proof,  and  has  succeeded  in  changing  one  into  the  other  by 
experimental  manipulation.  He  believes  that  the  acid-proof  con- 
dition has  some  bearing  upon  virulence,  and  speculates  that  the 
more  acid-proof  the  organisms  are,  the  less  virulent  they  will  be 
found. 

In  this  connection  the  work  of  Maher,f  who  claims  to  be  able, 
by  appropriate  methods  of  cultivation,  to  make  many  of  the  ordi- 
nary saprophytic  bacteria  (Bacillus  coli,  B.  subtilis,  etc.)  thor- 
oughly acid-proof,  must  be  mentioned. 

In  all  cases  where  the  detection  of  tubercle  bacilli  in  pus  or  secre- 
tions is  a  matter  of  clinical  importance,  it  must  be  remembered  that 
the  quantity  of  material  examined  by  the  staining  method  is  ex- 
tremely small,  so  that  a  few  bacilli  in  a  relatively  large  quantity  of 
matter  can  easily  escape  discovery. 

As  the  purpose  for  which  the  staining  is  most  frequently  performed 
is  the  differential  diagnosis  of  the  disease  through  the  demonstra- 
tion of  the  bacilli  in  sputum,  the  method  by  which  this  can  be 
accomplished  will  be  first  described. 

Staining  the  Bacillus  in  Sputum. — When  the  sputum  is  muco- 
purulent  and  nummular,  any  portion  of  it  may  suffice  for  ex- 
amination, but  if  the  patient  be  in  the  early  stages  of  tuberculosis, 
and  the  sputum  is  chiefly  thin,  seromucus,  and  flocculent,  care  must 
be  exercised  to  see  that  such  portion  of  it  as  is  most  likely  to  contain 
the  micro-organisms  be  examined. 

If  one  desires  to  make  a  very  careful  examination,  it  is  well  to 

*  "Berliner  klin.  Wochenschrift,"  April  6,  1908,  p.  691. 

t"  International  Conference  on  Tuberculosis,"  Philadelphia,  1907. 


66o  Tuberculosis 

have  the  patient  cleanse  the  mouth  thoroughly  upon  waking  in  the 
morning,  and  after  the  first  fit  of  coughing  expectorate  into  a  clean, 
wide-mouthed  bottle. 

The  best  result  will  be  secured  if  the  examination  be  made  on 
the  same  day,  for  if  the  bacilli  are  few  they  occur  most  plentifully 
in  small  flakes  of  caseous  matter,  which  are  easily  found  at  first, 
but  which  break  up  and  become  part  of  a  granular  sediment  that 
forms  in  decomposed  sputum. 

The  sputum  should  be  poured  into  a  watch-glass  and  held  over  a 
black  surface.  A  number  of  grayish-yellow,  irregular,  translucent 
fragments  somewhat  smaller  than  the  head  of  a  pin  can  usually 
be  found.  These  consist  principally  of  caseous  material  from  the 
tuberculous  tissue,  and  are  the  most  valuable  part  of  the  sputum 
for  examination.  One  of  the  fragments  is  picked  up  with  a  pointed 
match-stick  and  spread  over  the  surface  of  a  perfectly  clean  cover- 
glass  or  slide.  If  no  such  fragment  can  be  found,  the  purulent  part 
is  next  best  for  examination. 

The  material  spread  upon  the  glass  should  not  be  too  small  in 
amount.  Of  course,  a  massive,  thick  layer  will  become  opaque 
in  staining,  but  should  the  layer  spread  be,  as  is  often  advised, 
"as  thin  as  possible,"  there  may  be  so  few  bacilli  upon  the  glass 
that  they  are  found  with  difficulty. 

The  film  is  allowed  to  dry  thoroughly,  is  passed  three  times  through 
the  flame  for  fixation,  and  is  then  stained  and  examined. 

Where  examination  by  these  means  fails  to  reveal  the  presence 
of  bacilli  because  of  the  small  number  in  which  they  occur,  recourse 
may  be  had  to  the  use  of  caustic  potash  or,  what  is  better,  anti- 
formin  (g.v.)  for  digesting  the  sputum.  A  considerable  quantity 
of  sputum  is  collected,  receives  the  addition  of  an  equal  volume 
of  the  antiformin,  is  permitted  to  stand  until  the  formed  elements 
and  pus-corpuscles  have  been  dissolved,  is  then  shaken  and  poured 
into  centrifuge  tubes  and  whirled  for  fifteen  to  thirty  minutes. 
The  sediment  at  the  bottom  of  the  tubes  is  then  spread  upon  the 
glasses  and  stained  and  will  often  reveal  the  bacilli  which,  having 
been  freed  from  the  viscid  materials  in  the  sputum,  are  thrown 
down  in  masses  by  the  centrifuge. 

The  purpose  of  the  staining  being  the  discovery  of  the  tubercle 
bacillus,  success  is  only  possible  when  the  method  employed  en- 
ables that  particular  micro-organism  to  be  recognized,  as  such, 
so  soon  as  it  is  seen.  This  can  be  accomplished  by  taking  advantage 
of  the  "acid-proof"  quality  of  the  micro-organism,  which  permits 
it  to  take  up  the  penetrating  stains  employed,  but  does  not  permit 
it  to  let  them  go  again  in  the  bleaching  agents,  and  assume  the 
counter  stain.  It  is  owing  to  this  peculiarity  that  the  tubercle 
bacillus  alone  is  colored  blue  by  the  Koch-Ehrlich  method,  and  the 
tubercle  bacillus  alone  red  by  the  Ziehl  method,  and  it  is  because 
no  advantage  is  taken  of  the  acid-proof  peculiarity  in  using  Gram's 


Staining  66 1 

method,  that  the  latter,  which  colors  all  micro-organisms  stained, 
the  same  blue-black  color,  and  hence  is  not  differential,  is  never 
used  for  diagnostic  purposes. 

Ehrlich's  Method,  or  the  Koch-Ehrlich  Method. — Cover-glasses  thus  prepared  are 
floated,  smeared  side  down,  or  immersed,  smeared  side  up,  in  a  small  dish  of 
Ehrlich's  anilin- water  gentian  violet  solution: 

Anilin 4 

Saturated  alcoholic  solution  of  gentian  violet n 

Water 100 

and  kept  in  an  incubator  or  paraffin  oven  for  about  twenty-four  hours  at  about 
the  temperature  of  the  body.  Slides  upon  which  smears  have  been  made  can  be 
placed  in  Coplin  jars  containing  the  stain  and  stood  away  in  the  same  manner. 
When  removed  from  the  stain,  they  are  washed  momentarily  in  water,  and  then 
alternately  in  25  to  33  per  cent,  nitric  acid  and  60  per  cent,  alcohol,  until  the  blue 
color  of  the  gentian  violet  is  entirely  lost.  A  total  immersion  of  thirty  seconds  is 
enough  in  most  cases.  After  final  thorough  washing  in  60  per  cent,  alcohol,  the 
specimen  is  counterstained  in  a  dilute  aqueous  solution  of  Bismarck  brown  or 
vesuvin,  the  excess  of  stain  washed  off  in  water,  and  the  specimen  dried  and 
mounted  in  balsam.  The  tubercle  bacilli  are  colored  a  fine  dark  blue,  while  the 
pus-corpuscles,  epithelial  cells,  and  other  bacteria,  having  been  decolorized  by 
the  acid,  will  appear  brown. 

This  method,  requiring  twenty-four  hours  for  its  completion,  is  no  longer  used. 

ZiehVs  Method. — Among  clinicians,  Ziehl's  method  of  staining  with  carbol- 
fuchsin  has  met  with  just  favor.  It  is  as  follows:  After  having  been  spread, 
dried,  and  fixed,  the  cover-glass  is  held  in  the  bite  of  an  appropriate  forceps  (cover- 
glass  forceps),  or  the  slide  spread  at  one  end  is  held  by  the  other  end  as  a  handle, 
and  the  stain  (fuchsin,  i;  alcohol,  10;  5  per  cent,  phenol  in  water,  100)  dropped 
upon  it  from  a  pipet.  As  soon  as  the  entire  smear  is  covered  with  stain,  it  is  held 
over  the  flame  of  a  spirit  lamp  or  Bunsen  burner  until  the  stain  begins  to  vola- 
tilize a  little.  When  vapor  is  observed  the  heating  is  sufficient,  and  the  temper- 
ature can  be  maintained  by  intermittent  heating. 

If  evaporation  take  place,  a  ring  of  encrusted  stain  at  the  edge  prevents  the 
prompt  action  of  the  acid.  To  prevent  this,  more  stain  should  now  and  then  be 
added.  The  staining  is  complete  in  from  three  to  five  minutes,  after  which  the 
specimen  is  washed  off  with  water,  and  then  with  a  3  per  cent,  solution  of  hydro- 
chloric acid  in  70  per  cent,  alcohol,  25  per  cent,  aqueous  sulphuric,  or  33  per  cent, 
aqueous  nitric  acid  solution  dropped  upon  it  for  thirty  seconds,  or  until  the  red 
color  is  extinguished.  The  acid  is  carefully  washed  off  with  water,  the  specimen 
dried  and  mounted  in  Canada  balsam.  Nothing  will  be  colored  except  the  tuber- 
cle bacilli,  which  appear  red. 

Gobbet's  Method. — Gabbet  modified  the  method  by  adding  a  little  methylene 
blue  to  the  acid  solution,  which  he  makes  according  to  this  formula: 

Methylene  blue 2 

Sulphuric  acid 25 

Water 75 

In  Gabbet's  method,  after  staining  with  carbofc-fuchsin,  the  specimen  is  washed 
with  water,  acted  upon  by  the  methylene-blue  solution  for  thirty  seconds,  washed 
again  with  water  until  only  a  very  faint  blue  remains,  dried,  and  finally  mounted 
in  Canada  balsam.  The  tubercle  bacilli  are  colored  red;  the  pus-corpuscles, 
epithelial  cells,  and  unimportant  bacteria,  blue. 

Pappenheim,*  having  found  bacilli  stained  red  by  Ziehls'  method  in  the  sputum 
of  a  case  which  subsequent  postmortem  examination  showed  to  be  one  of  gan- 
grene of  the  lung  without  tuberculosis,  condemns  that  method  as  not  being 
sufficiently  differential,  and  recommends  the  following  as  superior  to  methods  in 
which  the  mineral  acids  are  employed: 

1.  Spread  the  film  as  usual. 

2.  Stain  with  carbol-fuchsin,  heating  to  the  point  of  steaming  for  a  few  minutes. 

3.  Pour  off  the  carbol-fuchsin  and  without  washing — 

*  "Berl.  klin.  Wochenschrift,"  1898,  No.  37,  p.  809. 


662  Tuberculosis 

4.  Dip  the  spread  from  three  to  five  times  in  the  following  solution,  allowing  it 

to  run  off  slowly  after  each  immersion: 

Corallin i  frm. 

Absolute  alcohol 100  cc. 

Methylene-blue ad  sat. 

Glycerin 20  cc. 

5.  Wash  quickly  in  water. 

6.  Dry. 

7.  Mount. 

The  entire  process  takes  about  three  minutes.  The  tubercle  bacilli  alone 
remain  red. 

Any  possible  relation  that  the  number  of  bacilli  in  the  expectora- 
tion of  consumptives  might  bear  to  the  progress  of  the  disease  was 
investigated  by  Nuttall.* 


,'/-«• 


V  s 

/ 


Fig.  271. — Bacillus  tuberculosis  in  sputum,  stained  with  carbolic  fuchsin  and 
aqueous  methylene-blue.     X  1000  (Ohlmacher). 

But  a  glance  down  the  columns  of  figures  in  the  original  article 
is  sufficient  to  show  that  the  number  of  bacilli  is  devoid  of  any 
practical  interest,  as  is  only  to  be  expected  when  one  considers  the 
pathology  of  the  disease  and  remembers  that  accident  may  cause 
wide  variations  in  the  quality,  if  not  in  the  quantity  of  the  sputum. 

Staining  the  Bacillus  in  Urine. — The  detection  of  tubercle  bacilli 
in  the  urine  is  sometimes  easy,  sometimes  difficult.  The  centrifuge 
should  be  used  and  the  collected  sediment  spread  upon  the  glass. 
li  there  be  no  pus  or  albumin  in  the  urine,  it  is  necessary  to  add  a 
little  white  of  egg  to  secure  good  fixation  of  the  urinary  sediment 
to  the  glass.  The  method  of  staining  is  the  same  as  that  for  sputum 

*"Bull.   of  the  Johns  Hopkins  Hospital,"   May  and  June,    1891,    n,   13. 


Isolation  663 

but  as  the  smegma  bacillus  (q.v.)  is  apt  to  be  present  in  the  urine, 
the  precaution  should  be  taken  to  use  Pappenheim's  solution 
for  differentiation  or  to  wash  the  stained  film  with  absolute  alcohol, 
that  it  may  be  decolorized  and  confusion  avoided. 

Staining  the  Bacillus  in  Feces. — It  is  difficult  to  find  tubercle 
bacilli  in  the  feces  because  of  the  relatively  small  number  of  bacilli 
and  large  bulk  of  feces. 

Staining  the  Bacillus  in  Sections  of  Tissue. — Ehrlich's  Method 
for  Sections. — Ehrlich's  method  must  be  recommended  as  the  most 
certain  and  best.  The  sections  of  tissue,  embedded  in  paraffin, 
should  be  cemented  to  the  slide  and  then  freed  from  the  embedding 
material. 

They  are  then  placed  in  the  stain  for  from  twelve  to  twenty-four  hours  and  kept 
at  a  temperature  of  37°C.  Upon  removal  they  are  allowed  to  lie  in  water  for 
about  ten  minutes.  The  washing  in  nitric  acid  (20  percent.)  which  follows  may 
have  to  be  continued  for  as  long  as  two  minutes.  Thorough  washing  in  60  per 
cent,  alcohol  follows,  after  which  the  sections  can  be  counterstained,  washed, 
dehydrated  in  96  per  cent,  and  absolute  alcohol,  cleared  in  zylol,  and  mounted  in 
Canada  balsam. 

Unna's  Method  for  Sections. — Unna's  method  is  as  follows:  The  sections  are 
placed  in  a  dish  of  twenty-four-hour-old,  newly  filtered  Ehrlich's  solution,  and 
allowed  to  remain  twelve  to  twenty-four  hours  at  the  room  temperature  or  one  to 
two  hours  in  the  incubator.  From  the  stain  they  are  placed  in  water,  where  they 
remain  for  about  ten  minutes  to  wash.  They  are  then  immersed  in  acid  (20  per 
cent,  nitric  acid)  for  about  two  minutes,  and  become  greenish  black.  From  the 
acid  they  are  placed  in  absolute  alcohol  and  gently  moved  to  and  fro  until  the 
pale-blue  color  returns.  They  are  then  washed  in  three  or  four  changes  of  clean 
water  until  they  become  almost  colorless,  and  then  removed  to  the  slide  by  means 
of  a  section-lifter.  The  water  is  absorbed  with  filter-paper,  and  then  the  slide  is 
heated  over  a  Bunsen  burner  until  the  section  becomes  shining,  when  it  receives  a 
drop  of  xylol  balsam  and  a  cover-glass. 

It  is  said  that  sections  stained  in  this  manner  do  not  fade  so  quickly  as  those 
stained  by  Ehrlich's  method. 

Gram's  Method. — The  tubercle  bacillus  stains  well  by  Gram's  method  and  by 
Weigert's  modification  of  it,  but  these  methods  are  not  adapted  for  differentiation. 
They  should  not  be  neglected  when  no  tubercle  bacilli  are  demonstrable  by  the 
other  methods,  as  they  are  particularly  well  adapted  to  the  demonstration  of  such 
of  the  organisms  as  may  not  be  acid-proof. 

Isolation. — Piatkowski*  has  suggested  that  the  cultivation  of  the 
tubercle  bacillus  and  other  "  acid-proof  "  organisms  may  be  achieved 
by  taking  advantage  of  their  ability  to  resist  the  action  of  formal- 
dehyd.  The  material  containing  the  acid-proof  organism  is  mixed 
thoroughly  with  10  cc.  of  water  or  bouillon,  which  receives  an  ad- 
dition of  2  or  3  drops  of  40  per  cent,  formaldehyd  or  "formalin." 
After  standing  from  fifteen  to  thirty  minutes  transfers  are  made  to 
appropriate  culture-media,  when  the  acid-proof  organisms  may 
develop,  the  others  having  been  destroyed  by  the  formaldehyd. 

Still  further  improvement  in  the  means  by  which  the  tubercle 
bacilli  can  be  secured  free  from  contamination  with  other  organisms 
and  from  surrounding  unnecessary  and  undesirable  materials,  has 
accrued  from  the  use  of  antiformin.  This  commercial  product, 
patented  in  1909  by  Axel  Sjoo  and  Tornell,  consists  of  Javelle  water 
*  "Deutsche  med.  Wochenschrift,"  June  9,  1904,  No.  23,  p.  878. 


664  Tuberculosis 

to  which  sodium  hydrate  is  added.  To  make  it  in  the  laboratory 
one  first  makes  the  Javelle  water  as  follows: 

K2C03 58 

CaO(OCl)2 80 

Water q.  s.  1000 

and  after  dissolving  the  salts  add  an  equal  volume  of  15  per  cent, 
aqueous  solution  of  caustic  soda. 

Uhlenhuth  and  Xylander*  investigated  its  usefulness  and  recom- 
mend it  highly  for  assisting  in  manipulating  the  tubercle  bacillus. 
The  sputum  or  tissue  supposed  to  contain  these  organisms  receives 
an  addition  of  antiformin,  by  which  the  tissue  elements,  the  pus  cells, 
the  mucous  and  other  objectionable  substances,  and  bacteria  are 
quickly  dissolved,  leaving  the  tubercle  bacilli  uninjured.  It  is 
then  centrifugalized,  the  fluid  poured  off  and  replaced  by  sterile  water 
or  salt  solution,  and  the  bacilli  washed,  after  which  they  are  again 
centrifugalized  and  caught  at  the  bottom  of  the  tube.  This  sedi- 
ment, rich  in  bacilli,  may  be  immediately  transferred  to  appropriate 
culture-media,  where  the  organisms  frequently  grow  quite  well, 
or  can  be  used  for  the  inoculation  of  guinea-pigs. 

The  most  certain  method  of  obtaining  a  culture  of  the  tubercle 
bacillus  from  sputum,  pus,  etc.,  is  to  first  inoculate  a  guinea-pig, 
allow  artificial  tuberculosis  to  develop,  and  then  make  cultures  from 
one  of  the  tuberculous  lesions. 

To  make  such  an  inoculation  with  material  such  as  sputum,  in 
which  there  are  many  associated  micro-organisms  that  may  destroy 
the  guinea-pig  from  septicemia,  Koch  advised  the  following  method, 
with  which  he  never  experienced  an  unfavorable  result. 

With  a  sharp-pointed  pair  of  scissors  a  snip  about  %  cm.  long  is 
made  in  the  skin  of  the  belly- wall.  Into  this  the  points  of  the  scissors 
are  thrust,  between  the  skin  and  the  muscles  for  at  least  i  cm.,  and 
the  scissors  opened  and  closed  so  as  to  make  a  broad  subcutaneous 
pocket.  Into  this  pocket  the  needle  of  the  hypodermic  syringe 
containing  the  injection,  or  the  slenden  glass  point  of  a  pipette  con- 
taining it,  is  introduced,  a  drop  of  fluid  expressed  and  gently  rubbed 
about  beneath  the  skin.  When  the  inoculating  instrument  is  with- 
drawn, the  mouth  of  the  pocket  is  left  open.  A  slight  suppuration 
usually  occurs  and  carries  out  the  organisms  of  wound  infection, 
while  the  tubercle  bacilli  are  detained  and  carried  to  the  inguinal 
nodes,  which  usually  enlarge  during  the  first  ten  days.  The  guinea- 
pigs  usually  die  about  the  twenty-first  day  after  infection. 

The  guinea-pig  is  permitted  to  live  until  examination  shows  the 
inguinal  glands  are  well  enlarged,  and  toward  the  middle  of  the  third 
week  is  chloroformed  to  death.  The  exterior  of  the  body  is  then 
wet  with  1:1000  solution  of  bichlorid  of  mercury  and  the  animal 
stretched  out,  belly  up,  and  tacked  to  a  board  or  tied  to  an  autopsy 

*  "  Arbeiten  a.  d.  Kaiserlichen  Gesundheilsamte,"  1909,  xxxi,  158;  "Centralbl. 
f.  Bakt.  u.  Parasitenk.,"  Referata,  1910,  XLV,  686. 


Cultivation 


665 


tray.  The  skin  is  ripped  up  and  turned  back.  The  exposed  ab- 
dominal muscles  are  now  washed  with  bichlorid  solution  and  a  piece 
of  gauze  wrung  out  of  the  solution  temporarily  laid  on  to  absorb  the 
excess.  With  fresh  sterile  forceps  and  scissors  the  abdominal  wall 
is  next  laid  open  and  fastened  back.  With  fresh  sterile  instruments 
the  spleen,  which  should  be  large  and  full  of  tubercles,  is  drawn 
forward  and,  one  after  another,  bits  the  size  of  a  pea  cut  or  torn  off 
and  immediately  dropped  upon  the  surface  of  appropriate  culture- 
media  in  appropriate  tubes.  The  fragments  of  tissue  from  the 
spleen  of  the  tuberculous  guinea-pig  are  not  crushed  or  comminuted, 
but  are  simply  laid  upon  the  undisturbed  surface  of  the  culture 
medium  and  then  incubated  for  several  weeks.  If  no  growth  is 
apparent  after  this  period,  the  bit  of 
tissue  is  stirred  about  a  little  and  the 
tube  returned  to  the  incubator,  where 
growth  almost  immediately  begins  from 
bacilli  scattered  over  the  surface  as  the 
bit  of  tissue  was  moved.  As  the  ap- 
propriate medium,  blood-serum  was 
recommended  by  Koch;  glycerin  agar- 
agar,  by  Roux  and  Nocard;  glycerinized 
potato,  by  Nocard;  coagulated  dogs' 
blood-serum,  by  Smith,  or  coagulated 
egg,  by  Dorset,  may  be  mentioned. 
The  most  certain  results  seem  to  follow 
the  employment  of  the  dogs'  serum 
and  egg  media. 

Cultivatio  n. — Blood-serum. — Koch 
first  achieved  artificial  cultivation  of  the 
tubercle  bacillus  upon  blood-serum, 
upon  which  the  bacilli  are  first  appa- 
rent to  the  naked  eye  in  about  two 
weeks,  in  the  form  of  small,  dry, 
whitish  flakes,  not  unlike  fragments  of 
chalk.  These  slowly  increase  in  size  at 
the  edges,  and  gradually  form  small 

scale-like  masses,  which  under  the  microscope  are  found  to  consist 
of  tangled  masses  of  bacilli,  many  of  which  are  in  a  condition  of 
involution.  The  medium  is  so  ill  adapted  to  the  requirements  of 
the  tubercle  bacillus  and  gives  such  uncertain  results  that  it  is  no 
longer  used. 

Glycerin  Agar-agar. — In  1887  Nocard  and  Roux*  gave  a  great 
impetus  to  investigations  upon  tuberculosis  by  the  discovery  that 
the  addition  of  from  4  to  8  per  cent,  of  glycerin  to  bouillon  and  agar- 
agar  made  them  suitable  for  the  development  of  the  bacillus,  and 
that  a  much  more  luxuriant  development  could  be  obtained  upon 
*  "Ann.  de  PInst.  Pasteur,"  1887,  No.  i. 


Fig.  272. — Bacillus  tuberculo- 
sis on  "glycerin  agar-agar." 


666 


Tuberculosis 


such  media  than  upon  blood-serum.  The  growth  upon  "  glycerin 
agar-agar"  resembles  that  upon  blood-serum.  A  critical  study  of 
the  relationship  of  massive  development  and  glycerin  was  made 
by  Kimla,  Poupe,  and  Vesley,*  who  found  that  the  most  luxuriant 

growth  occurred  when  the  culture-media  contained 

from  5  to  7  per  cent,  of  glycerin. 

Dogs'  Blood-serum. — A  very  successful  method  of 

isolating  the  tubercle  bacillus  has  been  published  by 

Smith.f 

A  dog  is  bled  from  the  femoral  artery,  the  blood  being  caught 
in  a  sterile  flask,  where  it  is  allowed  to  coagulate.  The  serum 
is  removed  with  a  sterile  pipette,  placed  in  sterile  tubes,  and 
coagulated  at  75°  to  j6°C.  Reichel  has  found  it  advantageous 
to  add  to  each  100  cc.  of  the  dogs'  serum  25  cc.  of  a  mixture 
of  glycerin  i  part,  and  distilled  water  4  parts.  The  whole  is 
then  carefully  shaken  without  making  a  froth,  and  dispensed 
in  tubes,  10  cc.  to  a  tube.  The  coagulation  and  sterilization 
he  effects  by  once  heating  to  gc0C.  for  three  to  five  hours.  At 
the  Henry  Phipps  Institute  in  Philadelphia  this  medium  was 
employed  with  thorough  satisfaction  for  the  isolation  of  many 
different  tubercle  bacilli.  Smith  prefers  to  use  a  test-tube  with 
a  ground  cap,  having  a  small  tubular  aperture  at  the  end,  in- 
stead of  the  ordinary  test-tube  with  the  cotton-plug.  The  pur- 
pose of  the  ground-glass  cap  is  to  prevent  the  contents  of  the 
tube  from  drying  during  the  necessarily  long  period  of  incuba- 
tion; that  of  the  tubulature,  to  permit  the  air  in  the  tubes  to 
enter  and  exit  during  the  contraction  and  expansion  resulting 
from  the  heating  incidental  to  sterilization. 

To  the  same  end  the  ventilators  of  the  incubator  are  closed, 
and  a  large  evaporating  dish  filled  with  water  is  stood  inside, 
so  that  the  atmosphere  may  be  constantly  saturated  with 
moisture. 

Egg  Media. — DorsetJ  recommends  an  egg  medium, 
which  has  the  advantage  of  being  cheap  and  easily 
prepared.  Eggs  are  always  at  hand,  and  can  be  made 
into  an  appropriate  medium  in  an  hour  or  two.  He 
also  claims  that  the  chemic  composition  of  the  eggs 
makes  them  particularly  adapted  for  the  purpose. 


Fig.  273-— 
Glass-capped 
culture  -tube 


the  isolation  of 
the  tubercle 
bacillus. 


The  medium  is  prepared  by  carefully  opening  the  egg  and 

used  by  Theo-     dropping  its  contents  into  a  wide-mouth  sterile  receptacle. 

bald  Smith  for  The  yolk  is  broken  with  a  sterile  wire  and  thoroughly  mixed 
with  the  white  by  gentle  shaking.  The  mixture  is  then  poured 
into  sterile  tubes,  about  10  cc.  in  each,  inclined  in  a  blood- 
serum  sterilizer,  and  sterilized  and  coagulated  at  7o°C.  on  two 
days,  the  temperature  being  maintained  for  four  or  five  hours 

each  day.     The  medium  appears  yellowish  and  is  usually  dry,  so  that  before 

using  it  is  well  to  add  a  few  drops  of  water. 

Potato. — Pawlowski§  was  able  to  isolate  the  bacillus  upon  potato. 
Sander  found  that  it  could  be  readily  grown  upon  various  vegetable 

*  "Revue  de  la  Tuberculose,"  1898,  vi,  p.  25. 

f  "Transactions  of  the  Association  of  American  Physicians,"  1898,  vol.  xin 
p.  417. 

t  "American  Medicine,"  1902,  vol.  in,  p.  555. 
§  "Ann.  de  1'Inst.  Pasteur,"  1888,  t.  vi. 


Cultivation 


667 


compounds,  especially  upon  acid  potato  mixed  with  glycerin 
Rosenau*  has  shown  that  it  can  grow  upon  almost  any  cooked  and 
glycerinized  vegetable  tissue. 

Animal  Tissues. — Frugonif  recommends 
that  the  tubercle  bacillus  be  isolated  and 
cultivated  upon  animal  tissue  and  organs 
used  as  culture-media.  He  especially 
recommends  rabbit's  lung  and  dog's  lung 
for  the  purpose.  The  tissues  are  first 
cooked  in  a  steam  sterilizer,  then  cut  into 
prisms,  placed  in  a  Roux  tube,  an  addition 
of  6  to  8  per  cent,  glycerin-water  added, 
so  as  to  bathe  the  lower  part  of  the  tissue 
and  keep  it  moist,  and  the  whole  then 
sterilized  in  the  autoclave. 

The  organisms  are  planted  upon  the 
tissue,  the  top  of  the  tube  closed  with  a 
rubber  cap,  and  the  culture  placed  in  the 
thermostat.  The  tubercle  bacilli  grow 
quickly  and  luxuriantly. 

Bouillon. — Upon  bouillon  to  which  6  per 
cent,  of  glycerin  has  been  added  the  bacillus 
grows  well,  provided  the  transplanted 
material  be  in  a  condition  to  float.  The 
organism  being  purely  aerobic  grows  only 
at  the  surface,  where  a  much  wrinkled, 
creamy  white,  brittle  pellicle  forms. 

Non-albuminous  Media. — 'Instead  of  re- 
quiring the  most  concentrated  albuminous 
media,  as  was  once  supposed,  Proskauer 
and  BeckJ  have  shown  that  the  organism 
can  be  made  to  grow  in  non-albuminous 
media  containing  asparagin,  and  that  it 
can  even  be  induced  to  grow  upon  a  mix- 
ture of  commercial  ammonium  carbonate, 
0.35  per  cent.;  primary  potassium  phos- 
phate, 0.15  per  cent.;  magnesium  sul- 
phate, 0.2  5  percent.;  glycerin,  1.5  percent. 
Tuberculin  was  produced  in  this  mixture. 

Gelatin. — -The  tubercle  bacillus  can  be        Fig.   274. — Bacillus  tu- 
grown  in  gelatin  to  which  glycerin  has     ^^Jef^fevefai 
been  added,  but  as  its  development  takes     months  old  (Curtis). 
place  only  at  37°  to  38°C.,  a  temperature 

at  which  gelatin  is  always  liquid,  its  use  for  the  purpose  has  no 
advantages. 

*  "Jour.  Amer.  Med.  Assoc.,"  1902. 

t  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  I.  Abl.  Orig.,  1910,  Lin,  553. 

J  "Zeitschrift  fur  Hygiene,"  Aug.  10,  1894,  xvm,  No.  i. 


668  Tuberculosis 

Appearance  of  the  Cultures.— Irrespective  of  the  media  upon 
which  they  are  grown,  cultures  of  the  tubercle  bacillus  present  certain 
characteristics  which  serve  to  separate  them  from  the  majority  of 
other  organisms,  though  insufficient  to  enable  one  to  identify  them 
with  certainty. 

The  bacterial  masses  make  their  appearance  very  slowly.  As  a 
rule  very  little  growth  can  be  observed  at  the  end  of  a  week,  and 
sometimes  a  month  must  elapse  before  the  growth  is  distinct. 

They  usually  develop  more  rapidly  upon  fluid  than  upon  solid 
media.  The  organism  is  purely  aerobic,  and  the  surface  growth 
formed  upon  liquids  closely  resembles  that  upon  solids. 


Fig.  275. — Bacillus  tuberculosis;  adhesion  cover-glass  preparation  from  a  four- 
teen-day-old blood-serum   culture.     X  100  (Frankel  and  Pfeiffer.) 

It  is  dry  and  lusterless,  coarsely  granular,  wrinkled,  slightly 
yellowish,  and  does  not  penetrate  into  the  substance  of  the  culture- 
medium.  It  sometimes  extends  over  the  surface  of  the  medium  and 
spreads  out  upon  the  contiguous  surface  of  moist  glass. 

When  the  medium  is  moist,  the  bacterial  mass  may  in  rare  in- 
stances be  shining  in  spots.  When  the  medium  is  dry,  it  is  apt  to 
be  scaly  and  almost  chalky  in  appearance. 

The  organism  grows  well  when  once  successfully  isolated,  and, 
when  once  accustomed  to  artificial  media,  not  only  lives  long  (six 
to  nine  months)  without  transplantation,  but  may  be  transplanted 
indefinitely. 

Reaction. — The  tubercle  bacillus  will  grow  upon  otherwise  ap- 
propriate media  whether  the  reaction  be  feebly  acid  or  feebly 
alkaline. 

Relation  to  Oxygen. — The  tubercle  bacillus  requires  oxygen,  and 
grows  only  upon  the  surface  of  the  culture-media. 


Pathogenesis  669 

Temperature  Sensitivity. — -The  bacillus  is  sensitive  to  tempera- 
ture variations,  not  growing  below  29°C.  or  above  42°C.  Rosenau* 
found  that  an  exposure  to  6o°C.  for  twenty  minutes  destroys  the 
infectiousness  of  the  tubercle  bacillus  for  guinea-pigs. 

Effect  of  Light. — It  does  not  develop  well  in  the  light,  and  when 
its  virulence  is  to  be  maintained  should  always  be  kept  in  the  dark. 
Sunlight  kills  it  in  from  a  few  minutes  to  several  hours,  according  to 
the  thickness  of  the  mass  of  bacilli  exposed  to  its  influence. 

Pathogenesis. — -Channels  of  Infection. — The  channels  by  which 
the  tubercle  bacillus  enters  the  body  are  numerous.  A  few  cases 
are  on  record  where  the  micro-organisms  have  passed  through  the 
placenta,  a  tuberculous  mother  infecting  her  unborn  child.  It  is  not 
impossible  that  the  passage  of  bacilli  through  the  placenta  in  this 


Fig.  276. — Bacillus  tuberculosis:   a,  Source,   human;   b,  source,  bovine.     Ma- 
ture colonies  on  glycerin-agar.     Actual  size  (Swithinbank  and  Newman.) 

manner  causes  the  rapid  development  of  tuberculosis  after  birth, 
the  disease  having  remained  latent  during  fetal  life,  for  Birch- 
Hirschfeld  has  shown  that  fragments  of  a  fetus,  itself  showing  no 
tuberculous  lesions,  but  coming  from  a  tuberculous  woman,  caused 
fatal  tuberculosis  in  guinea-pigs  into  which  they  were  inoculated. 

The  most  frequent  channel  of  infection  is  the  respiratory  tract, 
into  which  the  finely  pulverized  pulmonary  discharges  of  consump- 
tives and  the  dusts  of  infected  rooms  and  streets  enter.  Fliigge, 
Laschtschenko,  Heyman-Sticher,  and  Benindef  found  that  the 
greatest  danger  of  infection  was  from  the  atomized  secretions,  dis- 
charged during  cough,  from  the  tuberculous  respiratory  apparatus. 
Nearly  every  one  discharges  finely  pulverized  secretions  during 
coughing  and  sneezing,  as  can  easily  be  determined  by  holding  a 
mirror  before  the  face  at  the  time.  Even  though  discharged  by  con- 
sumptives, these  atoms  of  moisture  are  not  infectious  except  when 
there  are  open  lesions  in  the  lungs,  etc.  Experiment  showed 
that  they  usually  do  not  pass  farther  than  0.5  meter  from  the  patient, 
though  occasionally  they  may  be  driven  1.5  meters.  A  knowledge 


*  "Hygienic  Laboratory,"  Bulletin  No.  24,  Jan.,  1908. 
t"Zeitschrift  fur  Hygiene,"  etc.,  Bd.  xxx,  pp.  107,  125, 


139,  163,  193. 


670  Tuberculosis 

of  these  facts  teaches  us  that  visits  to  consumptives  should  not  be 
prolonged;  that  no  one  should  remain  continually  in  their  presence, 
nor  habitually  sit  within  2  meters  of  them;  also  that  patients  should 
always  hold  a  handkerchief  before  the  face  while  coughing.  The 
rooms  occupied  by  consumptives  should  also  be  frequently  washed 
with  a  disinfecting  solution. 

Probably  all  of  us  at  some  time  in  our  lives  inhale  living  virulent 
tubercle  bacilli,  yet  not  all  suffer  from  tuberculosis.  Personal 
variations  in  predisposition  seem  to  account  in  part  for  this,  as  it  has 
been  shown  that  without  the  formation  of  tubercles  virulent  bacilli 
may  sometimes  be  present  for  considerable  lengths  of  time  in  the 
bronchial  lymphatic  glands — -the  dumping-ground  of  the  pulmonary 
phagocytes. 

In  order  that  infection  shall  occur,  it  does  not  seem  necessary  that 
the  least  abrasion  or  laceration  shall  exist  in  the  mucous  lining  of 
the  respiratory  tract. 

Infection  also  commonly  takes  place  through  the  gastro-intestinal 
tract  from  infected  food.  Present  evidence  points  to  danger  from 
tubercle  bacilli  in  the  milk  of  cattle  affected  with  tuberculosis. 

The  ingested  bacilli  may  enter  the  tonsils  and  be  carried  to  the 
cervical  lymph-glands,  but  seem  more  commonly  to  reach  the  in- 
testine, from  which  they  enter  the  lymphatics,  sometimes  to  produce 
lesions  immediately  beneath  the  mucous  membrane,  sometimes 
to  invade  the  more  distant  mesenteric  lymphatic  glands,  but  more 
frequently  to  enter  the  thoracic  duct  and  then  through  the  venous 
system  find  their  way  to  the  lungs.  Passing  this  barrier  they  may 
distribute  through  the  arterial  systemic  circulation.  The  entrance 
of  tubercle  bacilli  into  the  systemic  circulation  with  subsequent 
deposition  in  the  brain,  bones,  joints,  etc.,  explains  primary  lesions 
of  these  tissues. 

Koch*  believed  that  human  beings  are  infected  only  by  bacilli 
from  other  human  beings,  and  his  paper  upon  this  subject  has 
stimulated  extensive  experimentation  on  the  problem.  Most 
authorities  believe  both  human  and  bovine  bacilli  to  be  equally 
infectious  for  man.  Behringf  believes  that  nearly  all  children  be- 
come infected  by  ingesting  tubercle  bacilli  in  milk,  though  a  certain 
predisposition  is  necessary  before  the  disease  can  develop.  Baum- 
garten  believes  that  all  children  harbor  bacilli  taken  in  the  food, 
but  that  the  disease  does  not  develop  until  a  certain  susceptibility 
occurs. 

Infection  also  occasionally  takes  place  through  the  sexual  appara- 
tus. In  sexual  intercourse  tubercle  bacilli  from  tuberculous  testicles 
can  enter  the  female  organs,  with  resulting  bacillary  implantation. 
Sexual  infections  are  usually  from  the  male  to  the  female,  primary 

*  "International  Congress  on  Tuberculosis,"  London,  1901,  and  Washington, 
1908. 

f  "Deutsche  med.  Wochenschrift,"  1903,  No.  39. 


Lesions  671 

tuberculosis  of  the  testicle  being  more  common  than  of  the  uterus 
or  ovaries. 

Wounds  are  also  occasional  avenues  of  entrance  for  tubercle 
bacilli.  Anatomic  tubercles  are  not  uncommon  upon  the  hands  of 
anatomists  and  pathologists,  most  of  these  growths  being  tuberculous 
in  nature.  Such  dermal  lesions  usually  contain  few  bacilli. 

Lesions. — The  macroscopic  lesions  of  tuberculosis  are  too  familiar 
to  require  a  description  of  any  considerable  length.  They  consist 
of  nodules,  or  collections  of  nodules,  called  tubercles,  irregularly 
scattered  through  the  tissues,  which  are  more  or  less  disorganized 
by  their  presence  and  retrogressive  changes. 

When  tubercle  bacilli  are  introduced  beneath  the  skin  of  a  guinea- 
pig,  the  animal  shows  no  sign  of  disease  for  a  week  or  two,  then  begins 
to  lose  appetite,  and  gradually  diminishes  in  flesh  and  weight.  Ex- 
amination usually  shows  a  nodule  at  the  point  of  inoculation  and 
enlargement  of  the  neighboring  lymphatic  glands.  The  atrophy 
increases,  the  animal  shows  a  febrile  reaction,  and  dies  at  the  end  of  a 
period  of  time  varying  from  three  to  six  weeks.  Post-mortem  ex- 
amination usually  shows  a  cluster  of  tubercles  at  the  point  of  inocu- 
lation, tuberculous  enlargement  of  lymphatic  glands  both  near  and 
remote  from  the  primary  lesion,  and  a  widespread  tuberculous  in- 
vasion of  the  lungs,  liver,  spleen,  peritoneum,  and  other  organs. 
Tubercle  bacilli  are  demonstrable  in  immense  numbers  in  all  the 
invaded  tissues.  The  disease  in  the  guinea-pig  is  usually  more 
widespread  than  in  other  animals  because  of  its  greater  susceptibility, 
and  the  death  of  the  animal  occurs  more  rapidly  for  the  same 
reason.  Intraperitoneal  injection  of  tubercle  bacilli  in  guinea-pigs 
causes  a  still  more  rapid  disease,  accompanied  by  widespread  lesions 
of  the  abdominal  organs.  The  animals  die  in  from  three  to  four 
weeks.  In  rabbits  the  disease  runs  a  longer  course  with  similar 
lesions.  In  cattle  and  sheep  the  infection  is  commonly  first  seen  in 
the  alimentary  apparatus  and  associated  organs,  and  may  be  limited 
to  them  though  primary  pulmonary  disease  also  occurs.  In  man 
the  disease  is  chiefly  pulmonary,  though  gastro-intestinal  and  general 
miliary  tuberculosis  are  common.  The  development  of  the  lesions 
in  whatever  tissue  or  animal  always  depends  upon  the  distribution 
of  the  bacilli  by  the  lymph  or  the  blood. 

The  experiments  of  Koch,  Prudden,  and  Hodenpyl,*  and  others 
have  shown  that  when  dead  tubercle  bacilli  are  injected  into  the 
subcutaneous  tissues  of  rabbits,  small  local  abscesses  develop  in 
the  course  of  a  couple  of  weeks,  showing  that  the  tubercle  bacilli 
possess  chemotactic  properties.  These  chemotactic  properties  seem 
to  depend  upon  some  other  irritant  than  that  by  which  the  chief 
lesions  of  tuberculosis  are  caused.  When  the  dead  tubercle  bacilli, 
instead  of  being  injected  en  masse  into  the  areolar  tissue,  are  intro- 
duced by  intravenous  injection  and  disseminate  themselves  singly 
*  "New  York  Med.  Jour.,"  June  6-20,  1891. 


672 


Tuberculosis 


or  in  small  groups,  the  result  is  quite  different,  and  the  lesions 
closely  resemble  those  caused  by  the  living  organisms. 

Baumgarten,  whose  researches  were  made  upon  the  iris,  found 
that  the  first  irritation  caused  by  the  bacillus  is  followed  by  multi- 
plication of  the  fixed  connective-tissue  cells  of  the  part.  The  cells 
increase  in  number  by  karyokinesis,  and  form  a  minute  cellular 
collection  or  primitive  tubercle. 

The  group  of  epithelioid  cells  and  lymphocytes  constituting 
the  primitive  tubercle  scarcely  reaches  visible  proportions  before 


»-:?£^^^Ws^^ 


Fig.  277. — Miliary  tubercle  of  the  testicle:  a,  Zone  of  epithelioid  cells  and 
leukocytes;  b,  area  of  coagulation-necrosis;  c,  giant  cell  with  its  processes;  per- 
ipherally arranged  nuclei  and  necrotic  center;  d,  seminiferous  tubule  (Cameron, 
in  "International  Text-book  of  Surgery"). 

central  coagulation-necrosis  begins.  The  cytoplasm  of  the  cells 
takes  on  a  hyaline  character;  the  chroma  tin  of  the  nuclei  becomes 
dissolved  in  the  nuclear  juice  and  gives  a  pale  but  homogeneous 
appearance  to  the  stained  nuclei.  As  the  tubercle  grows,  large 
protoplasmic  masses — giant  cells — which  contain  many  nuclei  are 
formed.  They  sometimes  occur  near  the  center,  more  frequently 
near  the  periphery  of  the  lesion. 

Giant  cells  are  not  always  formed  in  tubercles,  as  the  necrotic 
changes  are  sometimes  too  rapid  and  widespread. 

Tubercles  are  constantly  avascular — i.e.,  in  them  no  new  capillary 
blood-vessels  form — and  the  coagulation-necrosis  soon  destroys  pre- 


Lesions 


673 


existing  capillaries.  Avascularity  may  be  a  factor  in  the  necrosis 
of  the  larger  tuberculous  masses,  though  probably  playing  no 
important  part  in  the  degeneration  of  the  small  tubercles,  which  is 
purely  toxic. 


Fig.  278. — Tuberculosis  of  the  lung:  the  upper  lobe  shows  advanced  cheesy 
consolidation  with  cavity-formation,  bronchiectasis,  and  fibroid  changes;  the 
lower  lobe  retains  its  spongy  texture,  but  is  occupied  by  numerous  miliary 
tubercles. 

The  minute  primitive  tubercle  was  first  called  a  miliary  tubercle, 

and  small  aggregations  of  these,  "  crude  tubercles,"  by  Laennec. 

As   almost   all    tissues   contain   a   supporting    connective-tissue 

43 


674  Tuberculosis 

framework  whose  fibers  are  more  resistant  to  necrosis  than  the 
cells,  after  the  cells  of  a  tubercle  have  been  destroyed,  fibers  may 
still  be  visible  among  the  granules,  and  give  the  tubercle  a  reticulated 
appearance. 

As  a  rule,  tubercles  progressively  increase  in  size  by  the  inva- 
sion of  fresh  tissue.  The  tubercle  bacilli  are  usually  observed  in 
greatest  number  at  the  edges,  among  the  healthy  cells,  where  the 
nutrition  is  good.  From  this  position  they  are  swept  along  by 
currents  of  lymph  or  occasionally  are  picked  up  by  leukocytes  and 
transported  through  the  lymph-spaces,  until  the  phagocyte  falls  a 
prey  to  its  prisoner,  dies,  and  sows  the  seed  of  a  new  tubercle.  It  is 
by  such  continuous  invasion  of  new  tissue,  the  formation  of  necrotic 
areas  in  the  lungs,  and  evacuation  through  the  air-tubes  that  cavities 
are  formed.  In  pulmonary  tuberculosis  the  process  of  destruction 
is  greatly  accelerated  by  inspired  saprophytic  bacteria  that  live  in 
the  necrotic  tissue.  The  patient  also  suffers  from  secondary  infec- 
tions, especially  by  the  streptococcus  and  pneumococcus. 

If  the  vital  condition  of  the  individual  becomes  so  changed  that 
the  invasive  activity  of  the  bacilli  is  checked  or  their  death  brought 
about,  the  tubercle  begins  to  cicatrize,  and  becomes  surrounded  by 
a  zone  of  newly  formed  contracting  fibrillar  tissue,  by  which  it  is 
circumscribed  and  isolated.  This  constitutes  recovery  from 
tuberculosis.  Sometimes  the  process  of  repair  is  accomplished 
without  the  destruction  of  the  bacilli,  which  are  incarcerated  and 
retained.  Such  a  condition  is  called-  latent  tuberculosis,  and  may  at  a 
future  time  be  the  starting-point  of  a  new  infection. 

Virulence. — The  virulence  of  tubercle  bacilli  varies  considerably 
according  to  the  sources  from  which  they  are  obtained.  Bacilli 
from  different  cases  are  of  different  degrees  of  virulence,  and  bacilli 
from  different  animals  vary  still  more.  Lartigau,*  in  an  instructive 
paper  upon  "Variation  in  Virulence  of  the  Bacillus  Tuberculosis  in 
Man,"  found  much  variation  among  bacilli  secured  from  the  lesions 
of  human  tuberculosis.  The  virulent  was  tested  by  employing 
cultures  only  for  inoculation,  and  taking  of  each  bacillary  mass 
exactly  5  mg.  by  weight,  suspending  it  in  5  cc.  of  an  indifferent 
fluid  until  the  density  was  uniform  and  the  microscope  showed  no 
clumps,  and  injecting  into  rabbits  and  guinea-pigs,  pairs  of  animals 
being  injected  in  the  same  manner,  with  the  same  material,  at  the 
same  time,  and  being  subsequently  kept  under  similar  conditions. 
The  occurrence  of  tuberculosis  in  the  inoculated  animals  was  de- 
cided by  both  macroscopic  and  microscopic  tests. 

Lartigau  found  that  human  tubercle  bacilli  from  different  sources 
induced  varying  degrees  of  tuberculosis  in  animals;  that  the  in- 
jection of  the  same  culture  in  different  amounts  produces  different 
results;  that  the  extent  and  rapidity  of  development  usually  cor- 

*  "Journal  of  Medical  Research,"  July,  1901,  vol.  vi,  No.  i;  N.  S.,  vol.  i, 
No.  i,  p.  156. 


Chemistry  675 

respond  to  the  virulence  of  the  culture;  that  doses  of  i  mg.  of  a 
very  virulent  culture  may  induce  general  tuberculosis  in  rabbits 
in  a  very  short  time;  that  20  mg.  of  a  bacillus  of  low  virulence  may 
fail  to  produce  any  lesion  in  rabbits  or  guinea-pigs;  that  no  mor- 
phologic relationship  could  be  observed  between  the  bacilli  and  their 
virulence;  that  highly  virulent  bacilli  grew  scantily  on  culture- 
media  and  were  short  lived;  that  bacilli  of  widely  different  virulence 
may  be  present  in  any  one  of  the  various  human  tuberculous  lesions; 
that  in  scrofulous  lymphadenitis  the  bacilli  are  usually  of  low 
virulence;  the  bacilli  in  pulmonary  tuberculosis  with  ulceration  are 
of  feeble  virulence,  those  of  miliary  tuberculosis  of  very  great  viru- 
lence; that  the  so-called  " healed  tubercles"  of  the  lung  may  con- 
tain virulent  or  attenuated  bacilli;  that  individuals  suffering  from 
infection  with  a  bacillus  of  a  low  grade  of  virulence  may  be  again 
infected  with  extremely  virulent  tubercle  bacilli;  that  chronic 
tuberculosis  of  the  bones  may  contain  bacilli  of  high  or  low  virulence, 
and  that  variations  in  virulence  among  human  tubercle  bacilli 
may  possibly  sometimes  depend,  like  many  other  qualities  among 
tubercle  bacilli,  on  peculiarities  inherited  through  serial  trans- 
missions in  other  than  human  hosts. 

Chemistry  of  the  Tubercle  Bacillus. — Klebs*  found  that  the 
tubercle  bacillus  contains  two  fatty  bodies,  one  of  which,  having  a 
reddish  color  and  melting  at  42°C.,  can  be  extracted  with  ether. 
It  forms  about  20  per  cent,  by  weight  of  the  bacillary  substance. 
The  other  is  insoluble  in  ether,  t>ut  soluble  in  benzole,  with  which 
it  can  be  extracted.  It  melts  at  about  5o°C.  and  constitutes  1.14 
per  cent,  of  the  bacillary  substance.  After  removing  these  fatty 
bodies  the  bacilli  fail  to  resist  the  decolorant  action  of  acids  when 
stained  by  ordinary  methods,  so  that  it  seems  probable  that  their 
acid-resisting  power  depends  upon  them. 

De  Schweinitzf  showed  that  it  was  possible  to  extract  from 
the  tubercle  bacillus  an  acid  closely  resembling,  if  not  identical  with, 
teraconic  acid.  It  melts  at  161°  to  i64°C.  and  is  soluble  in  ether, 
water,  and  alcohol.  He  thinks  the  necrotic  changes  caused  by  the 
organism  depend  upon  it. 

RuppelJ  believes  that  three  different  fatty  substances  are  present 
in  the  tubercle  bacillus,  making  up  from  8  to  26  per  cent,  by  weight. 
The  first  can  be  extracted  with  cold  alcohol,  the  second  with  hot 
alcohol,  the  third  with  ether.  In  addition  to  the  fatty  substance 
Ruppel  also  found  what  he  believes  to  be  a  protamin,  and  calls 
tuber culosamin.  It  seems  to  be  combined  with  nucleinic  acid,  and, 
indeed,  from  it  he  isolated  an  acid  for  which  he  proposes  the  name 
tuberculinic  acid. 

*  "Centralbl.  f.  Bakt.,"  1896,  xx,  p.  488. 

t  "Trans.  Assoc.  of  Amer.  Phys.,"  1897;  "Centralbl.  f.  Bakt.,"  etc.,  Sept.  15, 
1897,  Bd.  xxn,  p.  200. 

J  "Zeitschrift  fur  physiol.  Chemie,"  1899,  xxvi. 


676  Tuberculosis 

Behring*  found  that  this  acid  contained  a  histon-like  body  whose 
removal  left  chemically  pure  tuberculinic  acid.  One  gram  of  this 
acid  is  capable  of  killing  a  6oo-gram  guinea-pig  when  administered 
beneath  the  skin.  One  gram  is  fatal  to  90,000  grams  of  guinea-, 
pig  when  introduced  into  the  brain.  If  injected  into  tuberculous 
guinea-pigs  it  is  much  more  fatal,  i  gram  destroying  60,000  when 
injected  subcutaneously  and  40,000,000  when  injected  into  the 
brain. 

Levenef  also  found  free  and  combined  nucleinic  acid  varying 
in  phosphorus  content  from  6.58  to  13.19  per  cent.  He  also  found 
a  glycogen-like  substance  that  reduced  Fehling's  solution  when 
heated  with  a  mineral  acid. 

Toxic  Products. — In  1890  Koch|  announced  some  observations 
upon  the  toxic  products  of  the  tubercle  bacillus  and  their  relation 
to  the  diagnosis  and  treatment  of  tuberculosis,  which  at  once  aroused 
an  enormous  though  transitory  enthusiasm.  The  observations  are, 
however,  of  great  importance.  Koch  found  that  when  guinea-pigs 
are  inoculated  with  tubercle  bacilli,  the  wound  ordinarily  heals 
readily,  and  soon  all  signs  of  local  disturbance  other  than  enlarge- 
ment of  the  lymphatic  glands  of  the  neighborhood  disappear.  In 
about  two  weeks,  however,  there  appears,  at  the  point  of  inocula- 
tion a  slight  induration,  which  develops  into  a  hard  nodule,  ulcer- 
ates, and  remains  until  the  death  of  the  animal.  If,  however,  in  a 
short  time  the  animals  be  reinoculated,  the  course  of  the  local 
lesion  is  changed,  and,  instead  of  healing,  the  wound  and  the  tissue 
surrounding  it  assume  a  dark  color,  become  obviously  necrotic,  and 
ultimately  slough  away,  leaving  an  ulcer  which  rapidly  and  per- 
manently heals  without  enlargement  of  the  lymph-glands. 

This  observation  was  made  by  injecting  cultures  of  the  living 
bacillus,  but  Koch  observed  that  the  same  changes  also  occur  when 
the  secondary  inoculation  is  made  with  killed  cultures  of  the  bacilli. 

It  was  also  observed  that  if  the  material  used  for  the  secondary 
injections  was  not  too  concentrated  and  the  injections  not  too  often 
repeated  (only  every  six  to  forty-eight  hours),  the  animals  treated 
improved  in  condition,  and  continued  to  live,  sometimes  (Pfuhl)  as 
long  as  nineteen  weeks. 

Tuberculin. — Koch  also  discovered  that  a  50  per  cent,  glycerin 
extract  of  cultures  of  the  tubercle  bacillus — tuberculin — produced 
the  same  effect  as  the  dead  cultures  originally  used,  and  announced 
the  discovery  of  this  substance  to  the  scientific  world,  in  the  hope 
that  the  prolongation  of  life  observed  to  follow  its  use  in  the  guinea- 
pig  might  also  be  true  of  man. 

The  active  substance  of  the  "tuberculin"  seems  to  be  an  al- 
buminous derivative  (bacterioprotein)  insoluble  in  absolute  alcohol. 

*  "Berliner  klin.  Wochenschrift,"  xxxvi. 

t  "  Jour,  of  Med.  Research,"  i,  1901. 

J  "Deutsche  med.  Wochenschrift,"  1891,  No.  343. 


Toxic  Products  677 

It  is  a  protein  substance  and  gives  all  the  characteristic  reactions. 
It  differs  from  the  toxalbumins  in  being  able  to  resist  exposure  to 
i2o°C.  for  hours  without  change.  Tuberculin  is  almost  harmless 
for  healthy  animals,  but  extremely  poisonous  for  tuberculous  ani- 
mals, its  injection  into  them  being  followed  either  by  a  violent 
febrile  reaction  or  by  death,  according  to  the  extent  of  the  dis- 
ease and  size  of  the  dose  administered. 

Preparation  of  Tuberculin. — The  preparation  of  tuberculin  is  simple.  Flasks 
made  broad  at  the  bottom  so  as  to  expose  a  considerable  surface  of  the  contained 
liquid  are  filled  to  a  depth  of  about  2  cm.  with  bouillon  containing  4  to  6  per  cent, 
of  glycerin,  and  preferably  made  with  veal  instead  of  beef  infusion.  They  are 
inoculated  with  pure  cultures  of  the  tubercle  bacillus,  care  being  taken  that  the 
bacillary  mass  floats  upon  the  surface,  and  are  kept  in  an  incubator  at  3'/°C.  In 
the  course  of  some  days  a  slight  surface  growth  becomes  apparent  about  the 
edges  of  the  floating  bacillary  mass,  which  in  the  course  of  time  develops  into  a 
firm,  coarsely  granular,  wrinkled  pellicle.  At  the  end  of  some  weeks  development 
ceases  and  the  pellicle  sinks,  a  new  growth  sometimes  occurring  from  floating 
scraps  of  the  original. 

Some  bacteriologists  prefer  to  use  small  Erlenmeyer  flasks  for  the  purpose,  but 
large  flasks,  which  contain  from  500  cc.  to  i  liter,  are  more  convenient.  The  con- 
tents of  a  number  of  flasks  of  well-grown  cultures  are  poured  into  a  large  porcelain 
evaporating  dish,  concentrated  over  a  water-bath  to  one-tenth  their  volume,  and 
filtered  through  a  Pasteur-Chamberland  filter.  This  is  crude  tuberculin. 

When  doses  of  a  fraction  of  a  cubic  centimeter  of  crude  tuberculin  are  injected 
into  tuberculous  animals,  an  inflammatory  and  febrile  reaction  occurs.  Superfi- 
cial tuberculous  lesions  (lupus)  sometimes  ulcerate  and  slough  away.  The  febrile 
reaction  is  sufficiently  characteristic  to  be  of  diagnostic  value,  though  tuberculin 
can  only  be  used  with  perfect  safety  as  a  diagnostic  agent  upon  the  lower  animals. 

From  the  "crude"  or  original  tuberculin  Koch  prepared  a  purified  or  "refined" 
tuberculin  by  adding  one  and  one-half  volumes  of  absolute  alcohol,  stirring 
thoroughly,  and  standing  aside  for  twenty-four  hours.  At  the  end  of  this  time  a 
flocculent  deposit  will  be  seen  at  the  bottom  of  the  vessel.  The  supernatant 
fluid  is  carefully  decanted  and  an  equal  volume  of  60  per  cent,  alcohol  poured  into 
the  vessel  for  the  purpose  of  washing  the  precipitate,  which  is  again  permitted  to 
settle,  the  fluid  decanted,  and  the  washing  thus  repeated  several  times,  after 
which  it  is  finally  washed  in  absolute  alcohol  and  dried  in  a  vacuum  exsiccator. 
The  white  powder  thus  prepared  is  fatal  to  tuberculous  guinea-pigs  in  doses  of  2  to 
10  mg.  It  is  soluble  in  water  and  glycerin  and  gives  the  protein  reactions. 
The  tuberculin  as  Koch  prepared  it  is  now  known  as  "concentrated"  or 
"Koch's  tuberculin,"  to  differentiate  it  from  the  "diluted  tuberculin"  some- 
times sold  in  the  shops,  which  is  the  same  thing  so  diluted  with  i  per  cent,  aqueous 
carbolic  acid  solution  that  i  cc.  equals  a  dose.  The  dose  of  the  concentrated 
tuberculin  is  0.4  to  0.5  cc.;  that  of  the  diluted  tuberculin,  i  cc. 

Tuberculin  does  not  exert  the  slightest  influence  upon  the  tubercle 
bacillus,  but  acts  upon  the  tuberculous  tissue,  augmenting  the 
poisonous  influence  upon  the  cells  surrounding  the  bacilli,  destroy- 
ing their  vitality,  and  removing  the  conditions  favorable  to  bacillary 
growth,  which  for  a  time  is  checked.  This  action  is  accompanied 
by  marked  hyperemia  of  the  perituberculous  tissue,  with  tran- 
sudation  of  serum,  softening  of  the  tuberculous  mass,  and  absorp- 
tion into  the  blood,  a  marked  febrile  reaction  resulting  from  the  in- 
toxication. 

Virchow,  who  well  understood  the  action  of  the  tuberculin,  soon 
showed  that  as  a  diagnostic  and  therapeutic  agent  in  man  its  use  was 
attended  by  grave  dangers.  The  destroyed  tissue  was  absorbed, 


6y8 


Tuberculosis 


but  with  it  some  of  the  bacilli,  which,  being  transported  to  new  tissue 
areas,  could  occasion  a  widespread  metastatic  invasion  of  the  disease. 
Old  tuberculous  lesions  which  had  been  encapsulated  were  sometimes 
softened  and  broken  down,  and  became  renewed  sources  of  infection 
to  the  individual,  so  that,  a  short  time  after  an  enthusiastic  recep- 
tion, tuberculin  was  placed  upon  its  proper  footing  as  an  agent 
valuable  for  diagnosis  in  veterinary  practice,  but  dangerous  in  human 
medicine,  except  in  cases  of  lupus  and  other  external  forms  of  tuber- 


Fig.  279. — Massive  culture  of  the  tubercle  bacillus  upon  the  surface  of  glycerin- 
bouillon,  used  in  the  manufacture  of  tuberculin. 

culosis  where  the  destroyed  tissue  could  be  readily  discharged  from 
the  surface  of  the  body. 

Many,  however,  continued  to  use  it,  and  Petruschky*  has  reported, 
with  careful  details,  22  cases  of  tuberculosis  which  he  claims  have 
been  cured  by  it. 

*  "Berliner  klin.  Wochenschrift,"  1899,  Dec.  18-25. 


Toxic  Products  679 

Recently  there  has  been  a  return  to  the  use  of  tuberculin  for  the 
diagnosis  of  tuberculosis,  it  being  claimed  that  by  the  use  of  minute 
doses,  several  times  repeated,  the  characteristic  reaction  and  a 
positive  diagnosis  can  be  obtained  without  danger. 

von  Pirquet*  found  that  if  a  drop  or  two  of  Koch's  (old)  tuberculin 
is  placed  upofi  the  skin  of  a  tuberculous  child,  and  a  small  scarifica- 
tion made  through  the  drop  with  a  sterile  lancet,  a  small  papule 
develops  at  the  point  of  inoculation  that  is  not  unlike  a  vaccine 
papule.  It  is  at  first  bright,  later  on  dark  red,  and  remains  for  a 
week.  Out  of  500  tests  made,  the  results  were  positive  in  nearly 
every  case  of  clinical  tuberculosis.  The  most  characteristic 
reactions  were  obtained  in  tuberculosis  of  the  bones  and  glands,  and 
the  method  is  recommended  chiefly  for  the  diagnosis  of  tuberculosis 
during  the  first  year  of  life.  This  method  of  testing  is  called  the 
"dermotuberculin  reaction" 

A  modification  of  this  method  by  Lignieresf  is  called  by  him  the 
"cutiluberculin  reaction"  Lignieres  soaps  and  shaves  the  skin  with  a 
safety  razor,  avoiding  scarification,  but  removing  the  superficial 
epidermal  cells  by  scraping,  and  then  applies  6  large  drops  of  un- 
diluted tuberculin,  rubbing  the  reagent  in  with  a  pledget  of  cotton. 
The  reaction  obtained  is  purely  local  and  without  fever. 

Moro  t  has  improved  upon  von  Pirquet's  method  by  using  the 
tuberculin  in  the  form  of  a  50  per  cent,  ointment  made  by  mixing 
equal  parts  of  "old  tuberculin"  and  lanolin,  which  is  rubbed  into  the 
skin  without  previous  scarification. 

Hiss§  says  that  "it  is  more  simple  and  equally  efficient  to  massage 
into  the  skin  a  drop  of  undiluted  'old  tuberculin.'" 

Calmettejj  suggested  the  ' ophthalmo-tuberculin  reaction"  which 
consists  of  instilling  i  drop  of  a  solution  of  prepared  tuberculin  into 
the  eye  of  the  suspect.  If  no  tuberculosis  exists,  no  reaction  follows, 
but  if  the  patient  be  infected  with  tuberculosis,  the  eye  becomes  red- 
dened in  a  few  hours  and  soon  shows  all  of  the  appearances  of  a  more 
or  less  pronounced  acute  mucopurulent  inflammation  of  the  con- 
junctiva. This  attains  its  maximum  in  six  or  seven  hours,  and  en- 
tirely recovers  in  three  days.  It  usually  causes  the  patient  very 
little  discomfort,  but  a  number  of  patients  have  been  unfortunate 
enough  to  suffer  from  supervening  corneal  ulceration  and  other  de- 
structive lesions  of  the  eye,  so  that  the  test  is  now  rarely  used, 
having  been  superseded  by  the  dermal  methods. 

The  method  of  preparing  the  solution  employed  by  Calmette 
is  to  precipitate  the  tuberculin  with  alcohol,  dry  the  precipi- 
tate and  dissolve  it  in  100  parts  of  distilled  water.  One  or  two 

*  "Ibid.,  May  20,  1907. 

t  "  Centralbl.  f .  Bakt.  u.  Parasitenk.,"  orig.,  XLVI,  Hft.  4,  March  10,  1908,  p. 

373- 

"Munch,  med.  Wochenschrift,"  1906,  p.  216. 
"Text-book  of  Bacteriology,"  1901,  p.  489. 
"La  Presse  M6dicale,"  June  19,  1907. 


68o  Tuberculosis 

drops  may  be  used.  Ordinary  tuberculin  must  be  avoided,  as 
the  glycerin  it  contains  causes  too  much  irritation  and  masks  the 
reaction. 

Priority  in  regard  to  the  theoretic  aspects  of  these  reactions 
seems  to  belong  to  Wolff-Eisner,*  who  was  the  first  to  point 
out  that  the  injection  of  all  albuminous  substances  resulted  in 
hypersensitivity  instead  of  immunity  unless  certain  precautions 
were  observed.  Upon  this  ground  Levyt  gives  him  credit  as  the 
founder  of  the  method.  The  reaction  is  undoubtedly  an  allergic 
phenomenon. 

KlebsJ  made  strong  claims  for  his  own  modifications  of  tuber- 
culin, known  as  antiphthisin  and  tuberculocidin,  but  according 
to  the  experimental  studies  of  Trudeau  and  Baldwin,  antiphthisin 
is  only  much  diluted  tuberculin,  and  exerts  no  demonstrable  in- 
fluence upon  the  tubercle  bacillus  in  vitro,  does  not  cure  tuberculosis 
in  guinea-pigs,  and  probably  inhibits  the  growth  of  the  tubercle 
bacillus  upon  culture-media  to  which  it  has  been  added  only  by  its 
acid  reaction. 

The  "bouillon-filtrate"  (bouillon  filtre),of  Denys§  is  a  porcelain 
filtrate  of  bouillon  culture  of  the  tubercle  bacillus  and  corresponds 
to  Koch's  original  tuberculin  before  concentration,  except  in  that 
it  has  not  been  subjected  to  heat. 

Tuberculin-R. — TR  or  tuberculin-R  appears  to  be  an  important 
addition  to  the  immunology  of  tuberculosis,  made  by  Koch.|| 

TR  signifies  "tuberkel  bacillen  resten"  or  bacillary  fragments. 

Pursuing  the  idea  of  fragmenting  the  bacilli,  or  treating  them  chemically  to 
increase  their  solubility,  Koch  found  that  a  10  per  cent,  sodium  hydrate  solution 
yielded  an  alkaline  extract  of  the  bacillus,  which,  when  injected  into  animals, 
produced  effects  similar  to  those  following  the  administration  of  tuberculin, 
except  that  they  were  more  brief  in  duration  and  more  constant  in  result;  but 
the  disadvantage  of  abscess  formation  following  the  injections  remained.  The 
fluid,  when  filtered,  possessed  the  properties  of  tuberculin. 

Mechanical  fragmentation  of  bacilli  had  been  employed  by  Klebs  in  his  studies 
of  antiphthisin  and  tuberculocidin,  and  Koch  now  used  it  with  advantage.  He 
pulverized  living,  virulent,  but  perfectly  dry  bacilli  in  an  agate  mortar,  in  order 
to  liberate  the  toxic  substance  from  its  protecting  envelope  of  fatty  acid,  triturat- 
ing only  very  small  quantities  of  the  bacteria  at  a  time. 

Having  thus  reduced  the  bacilli  to  fragments,  he  removed  them  from  the  mor- 
tar, placed  them  in  distilled  water,  washed  them,  and  collected  them  by  cen- 
trifugation,  as  a  muddy  residuum  at  the  bottom  of  an  opalescent,  clear  fluid. 
For  convenience  he  named  the  clear  fluid  TO;  the  sediment,  TR.  TO  was  found 
to  contain  tuberculin.  In  order  to  separate  the  essential  poison  of  the  bacteria 
as  perfectly  as  possible  from  the  irritating  tuberculin,  the  TR  fragments  were 
again  dried  perfectly,  triturated  once  more,  re-collected  in  fresh  distilled  water, 
and  recentrifugated.  After  the  second  centrifugation  microscopic  examination 
showed  that  the  bacillary  fragments  had  not  yet  been  resolved  into  a  uniform 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1904,  Orig.,  xxxvu. 
f  "Verein  fur  innere  Medizin  zu  Berlin,"  Dec.  16,  1907. 
J  "Die  Behandlung  der  Tuberculose  mit  Tuberculocidin,"  1892. 
§"Acad.  royale  de  med.  de  Belgique,"  Feb.  22,  1902;  abst.  "Centralbl.  f. 
Bakt.  u.  Parasitenk.,"  Ref.,  1902,  xxxi,  p.  563. 
||  "Deutsche  med.  Wochenschrift,"  1897,  No.  14. 


Toxic  Products  68 1 

mass,  for  when  TO  was  subjected  to  staining  with  carbol-fuchsin  and  methylene 
blue  it  was  found  to  exhibit  a  blue  reaction,  while  in  TR  a  cloudy  violet  reaction 
was  obtained. 

The  addition  of  50  per  cent,  of  glycerin  had  no  effect  upon  TO,  but  caused  a 
cloudy  white  deposit  to  be  thrown  down  from  TR.  This  last  reaction  showed 
that  TR  contained  fragments  of  the  bacilli  insoluble  in  glycerin. 

In  making  the  TR  preparation  Koch  advises  the  use  of  a  fresh,  highly  virulent 
culture  not  too  old.  It  must  be  perfectly  dried  in  a  vacuum  exsiccator,  and  the 
trituration,  in  order  to  be  thorough,  should  not  be  done  upon  more  than  100  mg. 
of  the  bacilli  at  a  time.  A  satisfactory  separation  of  the  TR  from  TO  is  said  to 
occur  only  when  the  perfectly  clear  TO  takes  up  at  least  50  per  cent,  of  the  solid 
substance,  as  otherwise  the  quantity  of  TO  in  the  final  preparation  is  so  great  as 
to  produce  undesirable  reactions. 

The  fluid  is  best  preserved  by  the  addition  of  20  per  cent,  of  glycerin,  which 
does  not  injure  the  TR  and  prevents  its  decomposition. 

The  finished  fluid  contains  10  mg.  of  solid  constituents  to  the  cubic  centimeter, 
and  before  administration  should  be  diluted  with  physiologic  salt  solution  (not 
solutions  of  carbolic  acid).  When  administering  the  remedy  to  man  the  injec- 
tions are  made  with  a  hypodermic  syringe  into  the  tissues  of  the  back.  The 
beginning  dose  is  ^500  mg->  rapidly  increased  to  20  mg.,  the  injections  being  made 
daily. 

Experiment  showed  that  TR  had  decided  immunizing  powers. 
Injected  into  tuberculous  animals  in  too  large  a  dose  it  produces 
a  reaction,  but  its  immunizing  effects  were  entirely  independent  of 
the  reaction.  Koch's  aim  in  using  this  preparation  in  the  therapeutic 
treatment  'of  tuberculosis  was  to  produce  immunity  against  the 
tubercle  bacillus  without  reactions  by  gradual  but  rapid  increase  of 
the  dose.  In  so  large  a  number  of  cases  did  Koch  produce  immunity 
to  tuberculosis  by  the  administration  of  TR,  that  he  believes  it 
proved  beyond  a  doubt  that  his  observations  are  correct. 

By  proper  administration  of  the  TR  he  was  able  to  render  guinea- 
pigs  so  completely  immune  that  they  were  able  to  withstand  inocula- 
tion with  virulent  bacilli.  The  point  of  inoculation  presents  no 
change  when  the  remedy  is  administered;  and  the  neighboring  lymph- 
glands  are  generally  normal,  or  when  slightly  swollen  contain  no 
bacilli. 

In  speaking  of  his  experiments  upon  guinea-pigs,  Koch  says: 

"I  have,  in  general,  got  the  impression  in  these  experiments  that  full  immuni- 
zation sets  in  two  or  three  weeks  after  the  use  of  large  doses.  A  cure  in  tubercu- 
lous guinea-pigs,  animals  in  which  the  disease  runs,  as  is  well  known,  a  very  rapid 
course,  may,  therefore,  take  place  only  when  the  treatment  is  introduced  early — 
as  early  as  one  or  two  weeks  after  the  infection  with  tuberculosis. 

"This  rule  avails  also  for  tuberculous  human  beings,  whose  treatment  must  not 
be  begun  too  late.  ...  A  patient  who  has  but  a  few  months  to  live  cannot 
expect  any  value  from  the  use  of  the  remedy,  and  it  will  be  of  little  use  to  treat  pa- 
tients who  suffer  chiefly  from  secondary  infection,  especially  with  the  streptococ- 
cus, and  in  whom  the  septic  process  has  put  the  tuberculosis  entirely  in  the 
background." 

One  very  serious  objection,  first  urged  against  commercially  pre- 
pared TR  by  Trudeau  and  Baldwin,*  is  that  it  is  possible  for  it  to 
contain  unpulverized,  and  hence  still  living,  virulent  tubercle  bacilli. 

*  "Medical  News,"  Aug.  28,  1897. 


682  Tuberculosis 

Thelling*  could  not  observe  any  good  effect  to  result  from  the  use 
of  Koch's  TR-tuberculin,  and,  like  Trudeau,  found  living,  virulent 
bacilli  in  the  preparation  secured  from  Hochst.  Many  others  have 
since  discovered  the  same  danger.  In  the  preparation  of  the  remedy 
it  will  be  remembered  that  no  antiseptic  or  germicide  was  added 
to  the  solutions  by  which  the  effects  of  accidental  failure  to 
crush  every  bacillus  could  be  overcome,  Koch  having  specially 
deprecated  such  additions  as  producing  destructive  changes  in  the 
TR.  Until  this  possibility  of  danger  can  be  removed,  and  our 
confidence  that  attempts  to  cure  patients  may  not  result  in  their 
infection  be  restored,  it  becomes  a  question  whether  TR  can  find 
a  place  in  human  medicine,  or  must  remain  an  interesting  labora- 
tory product. 

Baumgarten  and  Walzf  find  that  the  administration  of  tuber- 
culin-R  to  guinea-pigs  is  without  curative  effect.  They  insist 
that  the  results  obtained  are  like  those  of  the  old  tuberculin; 
that  "small  doses  are  of  no  advantage,  while  the  larger  the  doses 
one  employs,  the  greater  are  the  disadvantages  that  result  from 
their  employment." 

During  his  experiments  upon  the  agglutination  of  tubercle  bacilli, 
to  be  descrived  below,  Kochf  found  that  animals  injected  with  an 
emulsion  of  tubercle  bacilli  showed  great  increase  in  the  agglutinative 
power  of  the  blood.  This  led  him  to  suggest  that  a  new  preparation, 
"bacillary  emulsion"  Bazillenemulsion,  be  investigated  for  its  im- 
munizing and  curative  properties.  Many  are  still  using  it  and 
some  claim  good  results. 

It  is  almost  impossible  to  make  an  accurate  estimation  of  the 
usefulness  or  uselessness  of  therapeutic  preparations  of  tubercle 
bacilli  at  the  present  time,  not  only  because  of  their  diversity  of 
composition  and  the  enthusiasm  with  which  many  have  been 
exploited,  but  also  because  of  our  inability  to  compare  the 
results  attained  with  any  definite  standard.  The  advantages 
or  disadvantages  of  any  preparation,  therefore,  depend  upon 
the  personal  opinions  of  those  employing  them  rather  than  upon 
any  demonstration  regarding  them — a  very  unscientific  state  of 
knowledge. 

The  suggestion  of  A.  E.  Wright  that  the  administration  of  all  such 
products  should  be  controlled  by  an  examination  of  the  opsonic  power 
of  the  blood,  the  remedy  being  withheld  if  this  was  high  and  applied 
if  low,  the  utmost  care  being  taken  not  to  prolong  the  "negative 
phase,"  seemed  to  be  an  excellent  one,  affording  the  beginning  of  a 
scientific  method  of  studying  the  disease,  but  unfortunately  it  seems 
not  to  have  been  successful  in  practice,  and  the  tedium  and  expense 
of  the  examinations  makes  them  impracticable. 

*  "Centralbl.  f.  Bakt.,"  etc.,  July  5,  1902,  xxxn,  No.  i,  p.  28. 

t  "Centralbl.  f.  Bakt.  und  Parasitenk.,"  April  12,  1898,  xxm,  No.  14,  p.  593. 

j  "Deutsche  med.  Wochenschrift,"  1901,  No.  48,  p.  829. 


Antitubercle  Serums  683 

Agglutination. — Arloing*  and  Courmontf  found  it  possible  to 
prepare  homogenized  cultures  of  the  tubercle  bacillus,  and  saw  them 
agglutinated  by  the  serum  of  immunized  animals  and  by  the  serum 
of  tuberculous  patients.  The  subject  was  investigated  by  Koch,| 
who  carefully  reviewed  the  details  of  technic  and  investigated  the 
method,  which,  he  concluded,  was  valueless  for  the  diagnosis  of 
human  infection,  though  a  good  guide  to  the  extent  of  immunization 
achieved  by  the  therapeutic  administration  of  tuberculin-R.  Thel- 
ling§  has  also  shown  the  reaction  to  be  too  irregular  to  be  of  practical 
diagnostic  importance. 

The  technic  of  the  agglutination  test  as  given  by  Koch||  is  as 
follows : 

Any  culture  of  the  tubercle  bacillus  can  be  made  useful  by  the  following  treat- 
ment: Collect  the  bacillary  masses  upon  a  filter-paper  and  press  between  layers 
of  filter-paper  to  remove  the  fluid.  Weigh  out,  say,  0.2  gm.  of  the  solid  mass  and 
rub  it  in  an  agate  mortar,  adding,  drop  by  drop,  a  ^o  normal  sodium  hydroxid 
solution  until  the  proportion  of  i  part  of  the  culture  to  100  parts  of  the  solution  is 
reached. 

It  is  necessary  that  the  rubbing  be  thorough  in  order  that  the  firm  connection 
between  the  bacilli  shall  be  broken  up  and  the  organisms  distributed  throughout 
the  fluid.  The  operation  usually  lasts  fifteen  minutes.  The  fluid  is  then  placed 
in  a  hand  centrifuge  and  whirled  for  six  minutes,  then  pipetted  off,  and  rendered 
feebly  alkaline  by  adding  diluted  hydrochloric  acid  solution.  The  fluid  thus 
obtained  is  too  concentrated  to  be  used  in  this  form,  so  must  be  diluted  with  0.5 
per  cent,  carbolic  acid  in  0.85  per  cent,  sodium  chlorid  solution.  This  solution 
should  be  repeatedly  filtered  before  receiving  the  bacillary  suspension.  The 
quantity  of  bacillary  suspension  to  be  added  should  make  the  final  product  a  3000 
dilution  of  the  original.  It  should  look  like  water  by  transmitted  light,  but 
slightly  opalescent  by  reflected  light. 

The  serum  to  be  tested  is  added  in  proportions  of  i :  10,  i :  25,  i :  50,  i :  75,  i :  100, 
i:  200,  1:300,  etc.,  and  is  to  stand  for  twenty-four  hours.  By  inclining  the  tube 
and  looking  through  a  thin  stratum  of  the  fluid  the  agglutinations  can  be  at  once 
detected. 

Antitubercle  Serums. — Tizzoni  and  Centanni,**  Bernheim,tt 
Paquin,  J|  Viquerat§§  and  others  have  experimented  in  various  ways, 
hoping  that  the  principles  of  serum  therapy  might  apply  to  tuber- 
culosis. Nothing  has,  however,  been  achieved.  Maragliano's||  || 
antitubercle  serum  has  been  used  in  a  very  large  number  of  cases  in 
human  medicine,  but  the  glittering  results  reported  by  its  author 
have  not  been  confirmed.  Behring***  comments  upon  it  by  saying 
that  "  Maragliano's  tubercle  antitoxin  contains  no  antitoxin." 

*"  Congress   de   med.    int.  Montpellier,"  1898;  "Compt.  rendu  Acad.  de 
Sciences  de  Paris,"  1898,  T.  cxxvi,  pp.  1319-1321. 

t"  Compt.  rend.  Soc.  de  Biol.  de  Paris,"  1898,  No.  28,  v;  "Congr.  pour 
1' etude  de  la  Tuberculose,"  Paris,  1898. 

t  "Deutsche  med.  Wochenschrift,"  1901,  No.  48,  p.  829. 
§  Loc.  cit. 

||  "Deutsche  med.  Wochenschrift,"  1901,  No.  48,  p.  829. 
**  "Centralbl.  f.  Bakt.,"  etc.,  1892,  Bd.  xi,  p.  82. 
ft  "Ibid.,  1894,  Bd.  xv,  p.  654. 
tt  "New  York  Med.  Record,"  1895. 

§§  "Zur  Gewinnung  von  Antituberkulin,  Centralbl.  f.  Bakt.,"  etc.,  Nov.  5, 
1896,  xx,  Nos.  18,  19,  p.  674. 

Illl  "Berliner  klin.  Wochenschrift,"  1895,  No.  32. 
***  "Fortschritte  der  Med.,"  1897. 


684  Tuberculosis 

Babes  and  Proca,*  Maffucci  and  di  Vestea,f  McFarland,{  De 
Schweinitz,§  Fisch,||  and  Patterson**  have  all  endeavored  to  obtain 
serums  of  therapeutic  value  by  immunizing  animals  against  living 
or  dead  tubercle  bacilli  or  their  products,  but  without  success. 

From  these  discordant  observations,  the  more  favorable  of  which 
are  probably  the  hasty  records  of  inadequate  or  incomplete  experi- 
ments, the  conclusion  that  little  is  to  be  hoped  from  immune  serums 
in  the  treatment  of  tuberculosis  is  inevitable. 

Prophylaxis. — It  is  the  duty  of  every  physician  to  use  every  means 
in  his  power  to  prevent  the  spread  of  tuberculous  infection  in  the 
households  under  his  care.  To  this  end  patients  should  cease  to 
kiss  the  members  of  their  families  and  friends;  should  have  individual 
knives,  forks,  spoons,  cups,  napkins,  etc.,  carefully  kept  apart — 
secretly  if  the  patient  be  sensitive  upon  the  subject — from  those  of 
the  family,  and  scalded  after  each  meal;  should  have  their  napkins 
and  handkerchiefs,  as  well  as  whatever  clothing  or  bed-clothing  is 
soiled  by  them,  kept  apart  from  the  common  wash,  and  boiled;  and 
should  carefully  collect  the  expectoration  in  a  suitable  receptacle, 
that  is  sterilized  or  disinfected,  without  being  permitted  to  dry,  as 
it  has  been  shown  that  the  tubercle  bacillus  can  remain  alive  in  dried 
sputum  as  long  as  nine  months.  The  physician  should  also  give 
directions  for  disinfecting  the  bed-room  occupied  by  a  consumptive 
before  it  becomes  the  chamber  of  a  healthy  person,  though  this  should 
be  as  much  the  function  of  the  municipality  as  the  disinfection 
practised  after  scarlatina,  diphtheria,  and  smallpox. 

Boards  of  health  are  now  becoming  more  and  more  interested  in 
tuberculosis,  and,  though  exceedingly  slow  and  conservative  in  their 
movements,  are  disseminating  literature  with  the  hope  of  achieving 
by  volition  that  which  might  otherwise  be  regarded  as  cruel 
compulsion. 

So  long  as  tuberculosis  exists  among  men  or  cattle,  it  shows  that 
existing  hygienic  precautions  are  insufficient.  While  condemning 
any  unreasonable  isolation  of  patients,  we  should  favor  the  registra- 
tion of  tuberculous  cases  as  a  means  of  collecting  accurate  data  con- 
cerning their  origin;  insist  upon  the  careful  domestic  sterilization 
and  disinfection  of  all  articles  used  by  the  patients ;  recommend  public 
disinfection  of  the  houses  they  cease  to  occupy;  and  approve  of 
special  hospitals  for  as  many  (especially  of  the  poorer  classes, 
among  whom  hygienic  measures  are  almost  always  opposed)  as  can 
be  persuaded  to  occupy  them. 

*  "La  Med.  Moderne,"  1896,  p.  37. 
t  "Centralbl.  f.  Bakt.,"  etc.,  1896,  Bd.  xix,  p.  208. 
j  "Jour.  Amer.  Med.  Assoc.,"  Aug.  21,  1897. 

§"  Centralbl.  f.  Bakt.  und  Parasitenk.,"  Sept.   15,   1897,  Bd.  xxn,  Nos.  8- 
and  9. 

||  "Jour.  Amer.  Med.  Assoc.,"  Oct.  30,  1897. 
**  "Amer.  Medico-Surg.  Bull.,"  Jan.  25,  1898. 


Bovine  Tuberculosis  685 

BOVINE  TUBERCULOSIS 

BACILLUS  TUBERCULOSIS  Bovis 

The  tuberculous  diseases  of  the  lower  animals  and  especially  cattle 
have  lesions  closely  resembling  those  of  human  tuberculosis,  and 
containing  bacilli  similar  both  in  morphology  and  in  staining  reac- 
tion to  those  found  in  human  tuberculosis.  The  conclusion  that 
they  are  identical  seems  inevitable,  but  in  his  monograph  upon 
tuberculosis  Koch  called  attention  to  certain  morphologic  and  cul- 
tural differences  that  exist  between  bacilli  obtained  from  human  and 
from  animal  tuberculosis.  Unfortunately,  very  little  attention  was 
paid  to  the  subject  until  Theobald  Smith*  carefully  compared 
a  series  of  bacilli  obtained  from  human  sputum  with  another 
series  obtained  from  cattle,  horses,  hogs,  cats,  dogs,  and  other 
animals. 

His  observations  form  the  foundation  of  the  following  description 
of  the  bovine  tubercle  bacillus: 

Morphology. — 'The  size  of  the  bovine  bacillus  is  quite  constant, 
the  individuals  being  quite  short  (1-2  M).  They  are  straight,  not 
very  regular  in  outline,  and  sometimes  of  a  spindle,  sometimes  a 
barrel,  and  sometimes  an  oval  shape.  The  human  bacilli,  on  the 
other  hand,  are  prone  to  take  an  elongate  form  under  artificial 
cultivation. 

Staining. — 'The  bovine  bacillus  usually  stains  homogeneously;  the 
human  bacillus  commonly  shows  the  so-called  "beaded  appearance." 

Vegetation. — The  human  bacillus  grows  upon  dogs'  serum  much 
more  luxuriantly  and  rapidly  than  the  bovine  bacillus. 

Metabolic  Products. — Smith  f  observed  that  cultures  of  the  two 
organisms  in  glycerin  bouillon  differ  in  the  induced  reaction  of  the 
media.  The  cultures  of  the  bovine  bacillus  tend  toward  neutrality, 
those  of  the  human  bacillus  toward  acidity. 

Pathogenesis. — {a)  Guinea-pigs. — The  bovine  bacilli  are  more 
virulent  than  those  of  human  tuberculosis,  intraperitoneal  inocula- 
tion of  the  former  producing  death  in  adult  animals  in  from  seven 
to  sixteen  days;  of  the  latter,  in  from  ten  to  thirty-eight  days.  Sub- 
cutaneous inoculation  of  the  bovine  bacillus  causes  death  in  less 
than  fifty  days;  of  the  human  bacillus,  in  from  fifty  to  one  hundred 
days. 

(b)  Rabbits. — 'Rabbits  inoculated  into  the  ear  vein  with  the  bovine 
bacillus  die  in  from  seventeen  to  twenty-one  days.     Those  receiving 
human  bacilli  sometimes  live  several  months. 

(c)  Cattle. — Cows  and  heifers  receiving  intrapleural  and  intra- 
abdominal  injections  of  the  human  bacilli  usually  gain  in  weight  and 
show  no  symptoms.     When  examined  postmortem,  circumscribed 

*  "Trans.  Assoc.  Amer.  Phys.,"  1896,  xi,  p.  75,  and  1898,  xm,p.4i7;  "Jour, 
of  Experimental  Medicine,"  1898,  in,  495. 

t  "Trans.  Assoc.  Amer.  Phys.,"  1903,  vol.  xvin,  p.  109. 


686  Tuberculosis 

chronic  lesions  were  found.  Those  inoculated  with  the  bovine 
bacillus  lose  weight,  suffer  from  constitutional  symptoms,  and  show 
extensive  lesions  at  the  necropsy.  Two-thirds  of  the  cattle  inocu- 
lated experimentally  with  the  bovine  bacillus  die. 

Lesions. — In  general  the  lesions  produced  by  the  bovine  bacillus 
are  rapid,  extensive,  and  necrotic.  Many  bacilli  are  present. 
Those  produced  by  the  human  bacillus  are  more  apt  to  be  productive, 
chronic,  and  contain  relatively  few  bacilli.  The  bacilli  of  human 
tuberculosis  produce  lesions  with  many  giant  cells;  those  of  bovine 
tuberculosis,  lesions  with  rapid  coagulation  necrosis.  The  lesions 
resulting  from  the  intravenous  injection  of  human  bacilli  into  rabbits 
resembled  those  observed  by  Prudden  and  Hodenpyl*  after  the 
intravenous  injection  of  boiled,  washed  tubercle  bacilli. 

From  these  data  it  is  evident  that  the  bovine  bacillus  is  by  far 
the  more  virulent  and  dangerous  organism. 

At  the  International  Congress  on  Tuberculosis,  held  in  London, 
1901,  Koch  expressed  the  opinion  that  bovine  tuberculosis  was  not 
communicable  to  man.  The  matter  is  of  the  utmost  importance  to 
the  medical  profession  and  of  far-reaching  influence  upon  many  im- 
portant sanitary  measures  that  bear  directly  upon  the  public  health. 

Koch's  opinion,  being  opposed  to  all  that  had  been  believed  before, 
received  almost  universal  disapproval.  The  papers  by  Arloing,| 
Ravenel,J  and  Salmon  §  contain  evidence  showing  that  under  certain 
conditions  bovine  tuberculosis  can  be  communicated  to  man. 

Ravenel||  has  reported  3  cases  of  accidental  cutaneous  inoculation 
of  bovine  tuberculosis  in  man.  All  were  veterinary  surgeons  who 
became  infected  through  wounds  accidentally  inflicted  during  the 
performance  of  necropsies  upon  tuberculous  cattle.  The  tubercle 
bacilli  were  demonstrated  in  some  of  the  excised  cutaneous  nodules. 

Theobald  Smith,**  in  studying  3  cases  of  supposed  food  infection, 
found  what  corresponded  biologically  with  the  human  rather  than 
the  bovine  bacillus. 

In  a  later  paper  Kochff  analyzed  the  cases  usually  selected  from 
the  literature  to  prove  the  communicability  of  bovine  tuberculosis 
to  man,  and  showed  that  not  one  of  the  cases  really  proves  what  is 
claimed  for  it,  and  that  the  subject  requires  further  careful  investiga- 
tion and  demonstration  before  it  will  be  possible  to  express  any  posi- 
tive opinion  in  regard  to  it. 

During  the  years  that  have  elapsed  since  1901  and  the  present 
time  sentiment  has  been  almost  uniformly  against  Koch,  and  an 
enormous  literature  has  accumulated  that  in  reality  means  very 

*  "New  York  Med.  Jour.,"  June  6-20,  1891. 
t  "Lyon  Med.,"  Dec.i,  1901. 

j  "Univ.  of  Pa.  Bulletin,"  xiv,  p.  238,  1901;  "Lancet,"  Aug.  17  and  19,  1901; 
"Medicine,"  July  and  Aug.,  1902,  vol.  vm. 

§  "  Bull.  No.  33,  Bureau  of  Animal  Industry,"  U.  S.  Dept.  of  Agriculture,  1901. 
"Phila.  Med.  Jour.,"  July  21,  1900. 

1  "Amer.  Jour.  Med.  Sciences,"  Aug.,  1904,  vol.  cxxvm,  No.  389,  p.  216. 
ft  Eleventh  International  Congress  for  Tuberculosis,  Berlin,  1902. 


Bovine  Tuberculosis  687 

little.  The  most  important  is  that  of  the  Royal  Commission  on 
Tuberculosis  of  Great  Britain.*  The  general  tenor  of  this  report 
is  contrary  to  Koch's  views,  and  many  believed  it  settled  the  ques- 
tion. At  the  International  Congress  on  Tuberculosis  in  Washington, 
1908,  Koch  reviewed  the  subject  and  stated  his  continued  belief 
in  the  principle  he  had  enunciated  seven  years  before.  Practically 
the  same  contentions  were  raised  against  him  by  much  the  same 
group  of  men,  but  the  controversy  was  more  bitter  than  before. 
Koch,f  however,  leaves  us  in  no  doubt  upon  the  subject,  summarizing 
his  views  in  these  words: 

1.  The   tubercle  bacilli  of  bovine  tuberculosis  are  different  from   those  of 

human  tuberculosis. 

2.  Human  beings  may  be  infected  by  bovine  tubercle  bacilli,  but  serious  dis- 

eases from  this  cause  occur  very  rarely. 

3.  Preventive  measures  against  tuberculosis  should,   therefore,  be  directed 

primarily  against  the  propagation  of  human  tubercle  bacilli. 

He  weighed  the  contrary  evidence  that  had  been  collected  dur- 
ing seven  years,  showed  how  errors  had  crept  into  the  investi- 
gations, and  laid  down  certain  rules  to  be  observed  before  the 
experiments  could  be  accepted.  At  the  close  of  the  congress  the 
matter  remained  unsettled,  Koch  appearing  to  have  the  best  of 
the  argument. 

The  opponents  of  Koch  based  their  opinions  upon  the  supposed 
modifiability  of  the  tubercle  bacillus  in  different  environments. 
When  it  lived  in  man,  it  was  by  virtue  of  the  contact  with  the 
human  juices  and  their  chemical  peculiarities  compelled  to  assume 
the  human  form;  in  the  cow,  by  virtue  of  the  different  chemical 
conditions,  the  bovine  form,  etc.  Proofs  of  this  were,  however, 
wanting,  and  have  not  yet  been  published.  On  the  other  hand, 
MoriyaJ  seems  to  have  shown  that  such  changes  are  either  purely 
hypothetic  or  come  about  with  great  difficulty.  He  succeeded  in 
keeping  human  and  also  bovine  types  of  tubercle  bacilli  alive  in 
tortoises  for  twelve  months,  at  the  end  of  which  period  each  was 
found  unmodified  and  possessed  of  its  original  characteristics. 

It  was  Koch's  hope  to  be  able  to  finally  settle  the  whole  matter, 
and  to  this  end  he  asked  the  cooperation  of  many  laboratories 
throughout  different  parts  of  the  world.  Unfortunately  he  died 
before  the  results  could  be  compiled,  but  much  work  had  been  done 
and  much  support  thereby  given  his  views.  A  most  fertile  research, 
the  results  of  which  form  a  valuable  addition  to  our  knowledge  of 
the  problem  has  been  published  by  Park  and  Krumwiede,§  who, 
basing  their  opinions  upon  the  following  tabulation  of  1224  cases, 
come  to  the  following  conclusions: 

*  See  the  "British  Medical  Journal,"  1907  and  1908. 
t  "Jour.  Amer.  Med.  Assoc.,"  Oct.  10,  1908,  n,  No.  15,  p.  1256. 
:  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1909,  i,  Abt.  Orig.,  LI,  460. 
§  "Journal  of  Medical  Research,"  1910,  xxm,  No.  2,  p.  205;  1911,  xxv,  No.  2, 
P-  313- 


688 


Tuberculosis 


COMBINED  TABULATION  CASES  REPORTED  AND  OWN  SERIES  OF  CASES 


Diagnosis 

Adults  1  6  years 
and  over 

Children  5  to 
i  6  years 

Children  under 
5  years 

Human 

Bovine 

Human!  Bovine 

Human 

Bovine 

Pulmonary  tuberculosis  
Tuberculous  adenitis,  axillary  or  in- 
guinal 

644 

2 
27 
14 

6 
29 

5 

i 
27 
i? 
3 

2 

(I?) 

I 
4 

i 

i 
i 

i 

II 

4 
36 
8 

2 

4 

i 

7 
38 

2 

I 

21 

7 

3 

i 

3 

i 

36 

^3 

2 
15 

9 

13 
43 

3 

52 
27 
26 

i 
i 

I 

21 
13 

12 

5 
8 

i 
4 

Tuberculous  adenitis,  cervical  
Abdominal  tuberculosis.  
Generalized  tuberculosis,  alimentary 
origin          

Generalized  tuberculosis  
Generalized     tuberculosis    including 
meninges,  alimentary  origin  
Generalized     tuberculosis    including 
meninges              

Tubercular  meningitis              

Tuberculosis  of  bones  and  joints  
Genito-urinary  tuberculosis     

Tuberculosis  of  skin  

Miscellaneous  cases: 
Tuberculosis  of  tonsils  

Tuberculosis  of  mouth  and  cervical 
nodes         

Tuberculous  sinus  or  abscess  
Sepsis,  latent  bacilli  

Totals  

777 

10 

117 

215 

65 

Mixed  or  double  infections,  4  cases. 


Total  cases,  1224. 


Conclusions. — Bovine  tuberculosis  is  practically  a  negligible 
factor  in  adults.  It  very  rarely  causes  pulmonary  tuberculosis 
or  phthisis  which  causes  the  vast  majority  of  deaths  from  tuber- 
culosis in  man,  and  is  the  type  of  disease  responsible  for  the  spread 
of  the  virus  from  man  to  man. 

In  children,  however,  the  bovine  type  of  tubercle  bacillus  causes 
a  marked  percentage  of  the  cases  of  cervical  adenitis,  leading  to 
operation,  temporary  disablement,  discomfort,  and  disfigurement. 
It  causes  a  large  percentage  of  the  rarer  types  of  alimentary  tuber- 
culosis requiring  operative  interference  or  causing  the  death  of  the 
child  directly  or  as  a  contributing  cause  in  other  diseases. 

In  young  children  it  becomes  a  menace  to  life  and  causes  from 
6J£  to  10  per  cent,  of  the  total  fatalities  from  this  disease. 

Prophylaxis. — The  prevention  of  tuberculosis  in  cattle  is  a  matter 
of  vast  sanitary  importance.  Not  only  have  we  to  consider  the 
danger  of  infection  from  milk  containing  tubercle  bacilli,  but  also 
the  inferior  quality  and  diminished  usefulness  of  milk  and  flesh 


Bovine  Tuberculosis  689 

coming  from  animals  that  are  diseased.  The  extermination  of 
bovine  tuberculosis,  therefore,  becomes  imperative,  and  the  utmost 
efforts  should  be  made  to  bring  it  about.  Several  separate  meas- 
ures must  be  considered: 

1.  Improvement   in    the   methods   of   diagnosis,    by   which    the 
recognition  of  the  disease  is  made  possible  before  its  ravages  are 
great.     This  is  rapidly  coming  about  with  increasing  information 
regarding  the  use  and  abuse  of  tuberculin,  etc. 

2.  Means  by  which  infected  animals  shall  be  destroyed.     Here 
the  municipal  and  state  governments  furnish  inadequate  funds  to 
make  possible  the  destruction  of  diseased  cattle  without  adequate 
compensation — an  injustice  to  the  unfortunate  owner. 

3.  Means  of  preventing  the  infection  of  healthy  animals.     In 
many  places  this  is  being  achieved  with  brilliant  success  by  sep- 
aration of  the  herd,  healthy  and  newly  born  animals  constitut- 
ing one  part,  suspicious  animals  the  other.     By  these  means  valuable 
breeding  animals  can  be  kept  for  a  time,  at  least,  in  usefulness.     A 
second  and  less  successful  means  of  preventing  infection  is  by  means 
of   prophylactic   vaccination   of   the   healthy   animals    with    dead 
cultures,  modified  living  cultures,  or  by  bacteriotoxins  made  by 
comminuting  them. 

Experiments  of  this  kind  have  been  conducted  by  McFadyen,* 
on  a  large  scale  by  von  Behring,f  by  Pearson  and  Gilliland,|  Cal- 
mette  and  Guerin,§  and  by  Theobald  Smith,  ||  all  of  whom  think 
distinct  resisting  power  against  infection  by  the  tubercle  bacillus 
can  thus  be  brought  about. 

Tuberculin  Test  for  Tuberculosis  of  Cattle. — The  febrile  reac- 
tion caused  by  the  injection  of  tuberculin  into  tuberculous  animals 
is  an  important  adjunct  to  our  means  of  diagnosticating  the  disease. 
For  the  recognition  of  tuberculosis  in  cattle  it  is  easily  carried  out. 

To  make  a  satisfactory  diagnostic  test  the  temperature  of  the 
animal  should  be  taken  every  few  hours  for  a  day  or  two  before  the 
tuberculin  is  administered,  in  order  that  the  normal  diurnal  and 
nocturnal  variations  of  temperature  shall  be  known.  The  tuber- 
culin is  then  administered  by  hypodermic  injection  into  the  shoulder 
or  flank,  and  the  temperature  subsequently  taken  every  two  hours 
for  the  next  twenty-four  hours.  A  reaction  of  two  degrees  beyond 
that  normal  to  the  individual  animal  is  positive  of  tuberculosis.  After 
one  reaction  of  this  kind  the  animal  will  not  again  react  to  an  equal 
dose  of  tuberculin  for  a  number  of  weeks. 

*  "Jour.  Comp.  Path.  andTherap.,"  June,  1901. 

t  "Beitrage  zur  experimentellen  Therapie,"  1902,  Hft.  5. 

j"Jour.  of  Comp.  Med.  Vet.  Archiv,"  Nov.,  1902,  "Univ.  of  Penna.  Med. 
Bull.,"  April,  1905. 

§"Ann.  de  1'Inst.  Pasteur.,"  Oct.,  1905,  May,  1906,  and  July,  1907;  and 
"International  Congress  on  Tuberculosis,"  Washington,  1908. 

||  "Journal  of  Medical  Research,"  June,  1908,  xvm,  No.  3,  p.  451. 

44 


690  Tuberculosis 

FOWL  TUBERCULOSIS 

BACILLUS  TUBERCULOSIS  AVIUM 

The  occasional  spontaneous  occurrence  of  tuberculosis  in  chickens, 
parrots,  ducks,  and  other  birds,  observed  as  early  as  1868  by  RolofT* 
and  Paulicki,f  was  originally  attributed  to  Bacillus  tuberculosis 
hominis,  but  the  work  of  Rivolta,t  Mafucci,§  Cadio,  Gilbert  and 
Roger,  1 1  and  others  has  shown  that,  while  similar  to  it  in  many 
respects,  the  organism  found  in  the  avian  diseases  has  distinct  pe- 
culiarities which  make  it  a  different  variety,  if  not  a  separate  species. 
Cadio,  Gilbert,  and  Roger  succeeded  in  infecting  fowls  by  feeding 
them  upon  food  containing  tubercle  bacilli,  and  keeping  them  in 
cages  in  which  dust  containing  tubercle  bacilli  was  placed.  The 


Fig.  280. — Bacillus  tuberculosis  avium. 

infection  was  aided  by  lowering  the  temperature  of  the  birds  with 
antipyrin  and  lessening  their  vitality  by  starvation. 

Morphologic  Peculiarities. — Morphologically,  the  organism  found 
in  avian  tuberculosis  is  similar  to  that  found  in  the  mammalian 
disease,  but  is  a  little  longer  and  more  slender,  with  more  marked 
tendency  to  club  and  branched  forms.  Fragmented  and  beaded 
forms  occur  as  in  the  human  tubercle  bacilli. 

Staining. — The  avian  bacillus  stains  in  about  the  same  manner 
as  the  human  and  bovine  bacilli  and  has  an  equal  resistance  to 
the  decolorant  effect  of  acids. 

Cultivation. — Marked    rapidity    and    luxuriance  of  growth    are 

*  "Mag.  f.  d.  ges  Tierheilkunde,"  1868. 

"Beitr.  zur  vergl.  Anat.,"  Berlin,  1872. 
I  "Giorn.  anat.  fisiol.  e.  path.,"  Pisa,  1883. 
§  "Zeitschrift  fur  Hygiene,"  Bd.  xi. 
||  "La  Semaine  medicale,"  1890,  p.  45. 


Bacilli  Resembling  the  Tubercle  Bacillus  691 

characteristic  of  the  avian  bacillus,  which  grows  upon  ordinary 
agar-agar  and  bouillon  prepared  without  glycerin. 

The  growth  also  lacks  the  dry  quality  characteristic  of  cultures 
of  the  human  and  bovine  bacilli.  Old  cultures  of  the  bacillus  of 
fowl  tuberculosis  turn  slightly  yellow. 

Thermic  Sensitivity. — The  bacillus  also  differs  in  its  thermic 
sensitivity  and  will  grow  at  42°  to  45°C.  quite  as  well  as  at  379C., 
while  the  growth  of  the  human  and  mammalian  bacilli  ceases  at 
42°C.  Moreover,  growth  at  43°C.  does  not  attenuate  its  virulence. 
The  thermal  deathpoint  is  7o°C.  Upon  culture-media  it  is  said  to 
retain  its  virulence  as  long  as  two  years. 

Pathogenesis. — Birds  are  the  most  susceptible  animals  for 
experimental  inoculation,  the  embryos  and  young  being  more  sus- 
ceptible than  the  adults.  Artificial  inoculation  can  be  made  in  the 
subcutaneous  tissue,  in  the  trachea,  and  in  the  veins;  never  through 
the  intestine.  After  inoculation  the  birds  die  in  from  one  to  seven 
months.  The  chief  seat  of  the  disease  is  the  liver,  where  cellular 
(lymphocytic)  nodes,  lacking  the  central  coagulation  and  the  giant- 
cell  formation  of  mammalian  tuberculosis,  and  enormously  rich  in 
bacilli,  are  found.  The  disease  never  begins  in  the  lungs,  and  the 
fowls  that  are  diseased  never  show  bacilli  in  the  sputum  or  in  the 
dung. 

Guinea-pigs  are  quite  immune,  or  after  inoculation  develop  cheesy 
nodes,  but  do  not  die. 

Rabbits  are  easily  infected,  an  abscess  forming  at  the  seat  of 
inoculation,  nodules  forming  later  in  the  lungs,  so  that  the  dis- 
tribution is  quite  different  from  that  seen  in  birds.  It  is  possible 
that  the  avian  bacillus  occasionally  infects  man. 

The  possibility  that  this  bacillus  is  derived  from  the  same  stock 
as  the  tubercle  bacillus  is  strengthened  by  the  experiments  of 
Fermi  and  Salsano,*  who  succeeded  in  increasing  its  virulence  until 
it  became  fatal  to  guinea-pigs,  by  adding  glucose  and  lactic  acid  to 
the  cultures  inoculated. 

FISH  TUBERCULOSIS 

Dubarre  and  Terref  isolated  a  bacillus  having  the  tinctorial  and  morphologic 
characteristics  of  the  tubercle  bacillus  from  carp  suffering  from  a  tubercle-like 
affection.  In  respect  to  cultivation,  however,  it  was  unlike  the  tubercle  bacillus, 
growing  readily  upon  simple  culture-media  at  15°  to  3O°C.,  and  not  at  37°C. 

Weber  and  TaubeJ  found  the  same  organism,  or  what  seemed  to  be  the  same 
organism,  in  mud  and  in  a  healthy  frog. 

BACILLI  RESEMBLING  THE  TUBERCLE  BACILLUS 

It  is  not  improbable  that  the  bacilli  of  human,  bovine,  and  avian  tuberculosis 
are  closely  related  to  one  another,  and,  together  with  a  few  other  micro-organisms 
of  similar  morphology  and  staining  peculiarities,  have  a  common  ancestry 

*  "Centralbl.  f.  Bakt,"  etc.,  xn,  750. 

f  "Compt.  rendu  de  la  Soc.  de  Biol.  de  Paris,"  1897,  446... 

I  "Tuberkulose  Arbeiten  aus  dem  Kaiserlichen  Gesundheitsamte,"  1905. 


692  Tuberculosis 

and  are  descended  from  the  same  original  stock.  The  most  important  of  these 
similar  organisms  are  Bacillus  leprce  (q.v,),  B.  smegmatis,  and  Moeller's  grass 
bacillus. 

BACILLUS  SMEGMATIS 

Alvarez  and  Tavel,*  Matterstock,f  Klemperer  and  Bittu,J  Cowie,§  and 
others  have  described  peculiar  bacilli  in  smegma  taken  from  the  genitals  of  man 
and  the  lower  animals,  as  well  as  from  the  moist  skin  in  the  folds  of  the  groin, 
the  axillae,  and  the  anus.  They  are  also  sometimes  found  in  urine,  and 
occasionally  in  the  saliva  and  sputum. 

Morphology  and  Staining. — The  organisms  are  of  somewhat  variable  morph- 
ology, but  in  general  resemble  the  tubercle  bacillus,  stain  with  carbol-fuchsin,  as 
does  the  tubercle  bacillus,  and  resist  the  decolorant  action  of  acids.  They  are, 
however,  decolorized  by  absolute  alcohol,  though  Moeller  declares  the  smegma 
bacillus  to  be  absolutely  alcohol-proof  as  well  as  acid-proof,  and  admits  no  tinc- 
torial difference  between  it  and  the  tubercle  bacillus.  The  bacillus,  being  about 
the  size  and  shape  of  the  tubercle  bacillus,  is  very  readily  mistaken  for  it,  and  its 
presence  in  cases  of  suspected  tuberculosis  of  the  genito-urinary  apparatus,  and 
in  urine  and  other  secretions  in  which  it  is  likely  to  be  present,  may  lead  to  con- 
siderable confusion.  The  final  differentiation  may  have  to  rest  upon  animal 
inoculation. 

Cultivation. — The  cultivation  of  the  smegma  bacillus  is  difficult  and  was  first 
achieved  by  Czaplewski.||  Doutrelepont  and  Matterstock  cultivated  it  upon 
coagulated  hydrocele  fluid,  but  were  unable  to  transplant  the  growth  successfully. 

Novy**  recommends  the  cultivation  of  the  smegma  bacillus  by  inoculating  a 
tube  of  melted  agar-agar  cooled  to  5o°C.  with  the  appropriate  material,  and 
mixing  with  it  about  2  cc.  of  blood  withdrawn  from  a  vein  of  the  arm  with  a 
sterile  hypodermic  syringe.  The  blood-agar  mixture  is  poured  into  a  sterile 
Petri  dish  and  set  aside  for  a  day  or  two  at  37°C.  The  colonies  that  form  are  to 
be  examined  for  bacilli  that  resist  decolorization  with  acids. 

Moeller  ft  found  it  comparatively  easy  to  secure  cultures  of  the  smegma  bacillus 
by  a  peculiar  method.  To  secure  small  quantities  of  human  serum  for  the  pur- 
pose of  investigating  the  phenomena  of  agglutination  he  applied  small  cantharidal 
blisters  to  the  skins  of  various  healthy  and  other  men,  and  found  large  numbers  of 
acid-proof  bacilli  in  the  serum  saturated  with  epithelial  substance,  that  remained 
after  most  of  the  serum  had  been  withdrawn.  He  removed  the  skin  covering 
from  the  blister,  placed  it  in  the  remaining  serum,  and  kept  it  in  the  incubator  for 
three  or  four  days,  after  which  he  found  a  dry.  floating  scum,  which  consisted  of 
enormous  numbers  of  the  bacilli,  upon  the  serum.  From  this  growth  he  was 
subsequently  able  to  start  cultures  of  the  smegma  bacillus  upon  glycerin  agar- 
agar.  Human  blood-serum  is  thus  found  to  be  the  best  medium  upon  which  to 
start  the  culture. 

Agar. — A  culture  thus  isolated  grew  upon  all  the  usual  culture-media.  Upon 
glycerin-agar,  at  37°C.,  the  colonies  appeared  as  minute,  dull,  grayish- white,  dry, 
rounded  scales,  which  later  became  lobulated  and  velvety.  At  room  tempera- 
ture the  dry  appearance  of  the  growth  was  retained.  The  water  of  condensation 
remained  clear. 

Potato. — On  potato  the  growth  was -luxuriant,  grayish,  and  dull. 

Milk. — Milk  is  said  to  be  an  exceptionally  good  medium,  growth  taking  place 
in  it  with  rapidity  and  without  coagulation. 

Bouillon. — The  growth  forms  a  dry  white  scum  upon  the  surface,  the  medium 
remaining  clear. 

Pathogenesis. — So  far  as  is  known,  the  smegma  bacillus  is  a  harmless  sapro- 
phyte. 

*  "Archiv  de  Physiol.  norm,  et  Path.,"  1885,  No.  7. 

"Mittheil.  aus  d.  med.  Klin.  d.  Univ.  zu.  Wiirzburg,"  1885,  Bd.  vi. 
j  "Virchow's  Archives,"  v,  103. 

§  "Journal  of  Experimental  Medicine,"  1900-01,  vol.  v,  p.  205. 
||  "Miinchener  med.  Wochenschrift,"  1897. 
"*  "Laboratory  Work  in  Bacteriology,"  1899. 

ft  "  Centralbl.  f.  Bakt.  u.  Parasitenk,"  March  12,  1902,  (Originale),  Bd.xxxi, 
No.  7,  p.  278. 


Bacilli  Resembling  the  Tubercle  Bacillus  693 


MOELLER'S  GRASS  BACILLUS 

Bacilli  found  in  milk,  butter,  timothy  hay,  cow-dung,  etc.,  which  stain  like  the 
tubercle  bacillus  and  may  be  mistaken  for  it,  have  been  described  by  Moeller.* 
The  organisms  so  closely  resemble  the  tubercle  bacillus  that  guinea-pig  inocu- 
lations must  be  resorted  to  in  cases  of  doubt,  but  as  some  of  these  organisms 
sometimes  kill  the  guinea-pigs  after  a  month  or  two,  and  as  small  nodules  or 
tubercles  may  be  present  in  the  mesentery,  peritoneum,  liver,  lung,  etc.,  of  such 
animals,  the  diagnosis  may  have  to  be  subjected  to  the  further  confirmation  of  a 
histologic  examination  of  the  lesions  in  order  to  exclude  tuberculosis.  In  cases  of 
this  kind  it  should  not  be  forgotten  that  the  tubercle  bacillus  can  be  present  in  the 
substances  mentioned,  so  that  the  exact  differentiation  becomes  a  very  fine  one. 
An  instructive  study  of  these  organisms  has  been  made  by  Abbott  and  Gilder- 
sleeve,  f  who,  in  an  elaborate  work  upon  the  "Etiological  Significance  of  the 
Acid-resisting  Group  of  Bacteria,,  and  the  Evidence  in  Favor  of  Their  Botanical 
Relation  to  Bacillus  Tuberculosis,"  a  work  that  gives  complete  references  to  the 
literature  of  the  subject,  come  to  the  following  conclusions: 

1.  That  the  majority  of  the  acid-resisting  bacteria  may  be  distinguished  from 
true  tubercle  bacilli  by  their  inability  to  resist  decolorization  by  a  30  per  cent, 
solution  of  nitric  acid  in  water. 

2.  That  some  of  the  acid-resisting  bacteria  are  capable  of  causing  in  rabbits  and 
guinea-pigs  nodular  lesions  suggestive  of  tubercles;  that  these  lesions,  while  often 
very  much  like  tubercles  in  their  histologic  structure,  may  nevertheless  usually  be 
distinguished  from  them  by  the  following  peculiarities: 

(a)  When  occurring  as  a  result  of  intravenous  inoculation,  they  are  always 
seen  in  the  kidneys,  only  occasionally  in  the  lungs,  and  practically  not  at  all  in 
the  other  organs. 

(b)  They  constitute  a  localized  lesion,  having  no  tendency  to  dissemination, 
metastasis,  or  progressive  destruction  of  tissue  by  caseation. 

(c)  They  tend  to  terminate  in  suppuration  or  organization  rather  than  in  pro- 
gressive caseation,  as  is  the  case  with  true  tubercles. 

(d)  They  are  more  commonly  and  conspicuously  marked  by  the  actinomyces 
type  of  development  of  the  organisms  than  is  the  case  with  true  tubercles,  and 
these  actinomycetes  are  less  resistant  to  decolorization  by  strong  acid  solutions 
than  are  those  occasionally  seen  in  tubercles. 

3.  That  by  subcutaneous,  intravenous,  and  intrapulmonary  inoculation  of 
hogs  (4)  and  calves  (15)  the  typical  members  of  the  acid-resisting  group  are 
incapable  of  causing  lesions  in  any  way  suggestive  of  those  resulting  from  similar 
inoculations  of  the  same  animals  with  true  tubercle  bacilli. 

4.  That  though  occasionally  present  in  dairy  products,  they  are  to  be  regarded 
as  of  no  significance,  etiologically  speaking,  but  may  be  considered  as  accidental 
contaminations  from  the  surroundings,  and  not  as  evidence  of  disease  in  the 
animals. 

5.  That  the  designation  "bacillus"  as  applied  to  this  group  of  bacteria  and  to 
the  exciter  of  tuberculosis  is  a  misnomer;  they  are  more  correctly  classified  as 
actinomyces. 

THE  BUTTER  BACILLUS 

Petri,t  Rabinowitsch,§  and  Korn||  have  described,  as  Bacillus  butyricus,an 
acid-fast  organism  morphologically  like  the  tubercle  bacillus,  which  may  at  times 
be  found  in  butter.  Its  chief  importance  lies  in  the  confusion  that  may  arise 
through  mistaking  it  for  the  tubercle  bacillus  where  attention  is  paid  to  the  mor- 
phologic and  tinctorial  characters  only,  as  tubercle  bacilli  may  be  found  in  butter 
made  from  cream  from  the  milk  of  tuberculous  cattle. 

*  "Deutsche  med  Zeitung,"  1898,  p.  135;  "Deutsche  med.  Wochenschrift," 
1898,  p.  376,  etc. 

f  "Univ.  of  Penna.  Bulletin,"  June,  1902. 

j  "  Arbeiten  aus  dem  Kaiselichen  Gesundheitsamte,"  1897. 

§  "  Zeitschrif t  f iir  Hygiene,"  etc.,  1897. 

||  "Centralbl.  f.  Bakt.,"  etc.,  1899. 


694  Tuberculosis 

Isolation  and  cultivation  of  these  organisms  is  easy,  and  more  than  any  other 
measure  serves  to  differentiate  them  from  the  tubercle  bacillus,  as  they  grow  upon 
nearly  all  the  culture-media  with  rapidity  and  luxuriance. 

PSEUDOTUBERCULOSIS 

BACILLUS  PSEUDOTUBERCULOSIS 

Pfeiffer,*  Malassez  and  Vignal,f  Eberth,{  Chantemesse,§  Charrin,  and 
Roger||  have  all  reported  cases  of  so-called  pseudotuberculosis  occurring  in 
guinea-pigs,  and  characterized  by  the  formation  of  cellular  nodules  in  the  liver  and 
kidneys  much  resembling  miliary  tubercles.  Cultures  made  from  them  showed 
the  presence  of  a  small  motile  bacillus  which  could  easily  be  stained  by  ordinary 
methods.  When  introduced  subcutaneously  into  guinea-pigs,  the  original  disease 
was  reproduced. 

Morphology  and  Cultivation. — Bacillus  pseudotuberculosis  is  characterized  by 
Pfeiffer  as  follows:  The  organisms  are  rod-shaped,  the  rods  varying  in  length  (0.4 
to  1.2  ju)  and  sometimes  united  in  chains.  They  may  be  almost  round,  and  then 


p    0  » m<^t    *          *™"*       *    *^»  »*     ^ww         t*  ^  - 

^i^lpy 


Fig.  281. — Bacillus  pseudotuberculosis  from  agar-agar.     X  1000 
(Itzerott  and  Niemann.) 

resemble  diplococci.  They  stain  by  ordinary  methods,  but  not  by  Gram's 
method.  They  are  motile  and  have  flagella  like  the  typhoid  and  colon  bacilli. 
They  form  no  spores.  Upon  gelatin  and  agar-agar,  circular  colonies  with  a  dark 
nucleus  surrounded  by  a  transparent  zone  are  formed.  In  gelatin  punctures  the 
bacilli  grow  all  along  the  line  of  puncture  and  form  a  surface  growth  with  concen- 
tric markings.  The  gelatin  is  not  liquefied.  The  bacilli  grow  readily  upon  agar 
and  on  potato,  but  without  characteristic  appearances.  In  bouillon  a  diffuse 
turbidity  occurs,  with  floating  and  suspended  flakes.  Milk  is  not  altered. 

Pathogenesis. — The  bacillus  is  fatal  to  mice,  guinea-pigs,  rabbits,  hares,  and 
other  rodents  in  about  twenty  days  after  inoculation.  At  the  seat  of  inoculation 
an  abscess  develops,  the  neighboring  lymphatic  glands  enlarge  and  caseate,  and 
nodules  resembling  tubercles  form  in  the  internal  organs.  Similar  bacilli  studied 
by  Pfeiffer  were  isolated  from  a  horse  supposed  to  have  glanders. 


'Bacillare  tuberculose,  u.  s.  w.,"  Leipzig,  1889. 

'  Archiv  de  Physiol.  norm.  et.  Path.,"  1883  and  1884. 

Virchow's  Archiv,"  Bd.  en. 

Ann.  de  1'Inst.  Pasteur,"  1887. 

Compte-rendu  de  1'Acad.  des  Sci.,"  Paris,  t.  cvi. 


CHAPTER  XXX 
LEPROSY 

BACILLUS  LEPR^E  (HANSEN)* 

General  Characteristics. — A  non-motile,  non-flagellate,  non-sporogenous, 
chromogenic,  non-liquefying,  non-aerogenic,  distinctly  aerobic,  parasitic  and 
highly  pathogenic,  acid-resisting  bacillus,  staining  by  Gram's  method,  and  culti- 
vable upon  specially  prepared  artificial  media.  It  does  not  form  indol,  or  acidu- 
late or  coagulate  milk. 

Leprosy  very  early  received  attention  and  study.  Moses  in- 
cluded in  the  laws  to  the  people  of  Israel  rules  for  its  diagnosis,  for 
the  isolation  of  the  sufferers,  for  the  determination  of  recovery,  and 
for  the  sacrificial  observances  to  be  fulfilled  before  the  convalescent 
could  once  more  mingle  with  his  people.  The  Bible  is  replete  with 
miracles  wrought  upon  lepers,  and  during  the  times  of  biblical 
tradition  it  seems  to  have  been  an  exceedingly  common  and  malig- 
nant disease.  Many  of  the  diseases  called  leprosy  in  the  Bible  were, 
however,  in  all  probability,  less  important  parasitic  skin  affections. 

Distribution. — At  the  present  time,  although  we  hear  very  little 
about  it  in  the  northern  United  States,  leprosy  is  a  widespread  dis- 
ease and  exists  much  the  same  as  it  did  several  thousand  years  ago 
in  Palestine,  Syria,  Egypt,  and  the  adjacent  countries,  and  is 
common  in  China,  Japan,  and  India.  South  Africa  has  many 
cases,  and  Europe,  especially  Norway,  Sweden,  and  parts  of  the 
Mediterranean  coast,  a  considerable  number.  In  certain  islands, 
especially  the  Sandwich  and  Philippine  Islands,  it  is  endemic.  In 
the  United  States  the  disease  is  uncommon,  the  Southern  States 
and  Gulf  coast  being  chiefly  affected. 

A  commission  of  the  Marine-Hospital  Service,  formed  for  the 
purpose  of  investigating  the  prevalence  of  leprosy,  in  1902  re- 
ported 278  existing  cases  in  the  United  States.  Of  these,  155 
occurred  in  the  State  of  Louisiana.  The  other  States  with  numerous 
cases  were  California,  24;  Florida,  24;  Minnesota,  20;  and  North 
Dakota,  16.  No  other  State  had  more  than  7  (New  York).  Of  the 
cases,  145  were  American  born,  120  foreign  born,  the  remainder 
uncertain. 

Etiology.— The  cause  of  leprosy  is,  without  doubt,  the  lepra 
bacillus,  discovered  by  Hansen  in  1879. 

Morphology. — The  bacillus  is  about  the  same  size  as  the  tubercle 
bacillus.     Its  protoplasm  commonly  presents  open  spaces  of  frac- 
*  "Virchow's  Archives,"  1879. 
695 


696  Leprosy 

tures,  giving  it  a  beaded  appearance,  like  the  tubercle  bacillus.  It 
occurs  singly  or  in  irregular  groups.  There  is  no  characteristic 
grouping  and  filaments  are  unknown.  It  is  not  motil^  and  has  no 
flagella  and  no  spores. 

Duval  found  that  the  cultivated  bacilli  are  longer,  more  curved, 
and  show  a  greater  irregularity  in  the  distribution  of  the  chromatin 
than  those  in  the  tissues  where  they  are  short,  slender,  and  slightly 
curved.  In  artificial  cultures  there  is  a  delicate  filamentous  ar- 
rangement of  the  bacilli,  especially  where  they  have  become  ac- 
customed to  a  saprophytic  existence.  They  often  contain  distinct 
metachromatic  granules  analogous  to  those  met  with  in  certain 
forms  of  the  diphtheria  bacillus.  They  are  quite  pleomorphous, 
and  in  the  same  culture  all  forms  occur,  from  solidly  staining  coccoid 


Fig.  282. — Lepra  bacilli.     Smear  from  a  lepra  node  stained  with  carbol-fuchsin 
(Kolle  and  Wassermann). 

shapes  to  slender  slightly  curved  filaments,  with  numerous  chromatic 
segments  and  occasional  metachromatic  granules.  Sometimes 
the  organisms  are  pointed  at  the  ends. 

Czaplewski  found  that  the  lepra  bacilli  in  his  cultures  colored 
uniformly  when  young,  but  were  invariably  granular  when  old.  The 
more  rapidly  the  organism  grew,  the  more  slender  it  appeared. 

Staining. — -It  stains  in  very  much  the  same  way  as  the  tubercle 
bacillus,  but  permits  of  a  more  ready  penetration  of  the  stain,  so 
that  the  ordinary  aqueous  solutions  of  the  anilin  dyes  color  it  quite 
readily.  The  property  of  retaining  the  color  in  the  presence  of 
the  mineral  acids  also  characterizes  the  lepra  bacillus,  and  the 
methods  of  Ehrlich,  Gabbet,  and  Unna  for  staining  the  tubercle 
bacillus  can  be  used  for  its  detection.  It  stains  well  by  Gram's 
method  and  by  Weigert's  modification  of  it,  by  which  beautiful 
tissue  specimens  can  be  prepared. 

Cultivation. — Many    endeavors    have    been    made    to    cultivate 


Cultivation 


697 


this  bacillus  upon  artificially  prepared  media,  but  in  1903  Hansen,* 
who  discovered  the  organism,  declared  that  no  one  had  yet  culti- 
vated it. 

Bordoni-Uffreduzzif  was  able  to  cultivate  a  bacillus  which  par- 
took of  the  staining  peculiarities  of  the  lepra  bacillus  as  it  appears 
in  the  tissues,  but  differed  in  morphology. 

Czaplewskit    confirmed    the   work    of    Bordoni-Uffredozzi,    and 


L/SVi  V  ^,>'  '"  ~* -ijs^ 

^  CB-V;*-^?    (S  ^       ^ 


r  ;;Bi 


w*<x&Jfm 
^HfeP*   01 


Fig.  283. — Section  of  one  of  the  nodules  from  the  patient  shown  in  Fig.  285, 
stained  by  the  Weigert-Gram  method  to  show  the  lepra  bacilli  scattered  through 
the  tissue  and  inclosed  in  the  large  vacuolated  "lepra-cells."  Magnified  1000 
diameters. 

described  a  bacillus  supposed  to  be  the  lepra  bacillus,  which  he 
succeeded  in  cultivating  from  the  nasal  secretions  of  a  leper. 

The  bacillus  was  isolated  upon  a  culture-medium  consisting  of 
glycerinized  serum  without  the  addition  of  salt,  peptone,  or  sugar. 
The  mixture  was  poured  into  Petri  dishes,  coagulated  by  heat,  and 
sterilized  by  the  intermittent  method. 

*  Kolle  and  Wassermann's  "Handbuch  der  pathogenen  Mikroorganismen," 
n,  p.  184,  1903. 

t  "Zeitschrift  1.  Hygiene,"  etc.,  1884,  in. 

t  "Centralbl.  f.  Bakt.  und  Parasitenk.,"  Jan.  31,  1898,  vol.  xxm,  Nos.  3  and 
4,  p.  97. 


698  Leprosy 

The  secretion,  being  rich  in  lepra  bacilli,  was  taken  up  with 
a  platinum  wire  and  inoculated  upon  the  culture-medium  by  a 
series  of  linear  strokes.  The  dishes  were  then  sealed  with  paraffin 
and  kept  in  the  incubating  oven  at  37°C. 

Numerous  colonies,  chiefly  of  Staphylococcus  aureus  and  the 
bacillus  of  Friedlander,  developed,  and  in  addition  a  number  of 
colonies,  composed  of  slender  bacilli  about  the  size  and  form  of 
the  lepra  bacillus. 

These  colonies  were  grayish  yellow,  humped  in  the  middle,  i  to 
2  mm.  in  diameter,  irregularly  rounded,  and  uneven  at  the  edges. 
They  were  firm  and  could  be  entirely  inverted  with  the  platinum 
wire,  although  the  consistence  was  crumbly.  They  were  excavated 
on  the  under  side. 

The  colonies  that  formed  upon  agar-agar  were  much  like  those 
described  by  Bordoni-Uffreduzzi,  and  appeared  as  isolated,  grayish, 
rounded  flakes,  thicker  in  the  center  than  at  the  edges,  and  char- 
acterized by  an  irregular  serrated  border  from  which  a  fine  irregular 
network  extended  upon  the  medium.  These  projections  consisted 
of  bundles  of  the  bacilli. 

When  a  transfer  was  made  from  one  of  these  colonies  to  fresh 
media,  the  growth  became  apparent  in  a  few  days  and  assumed  a 
band-like  form,  with  a  plateau-like  elevation  in  the  center. 

The  bacillus  thus  isolated  grew  with  moderate  rapidity  upon 
all  the  ordinary  culture-media  except  potato.  Upon  blood-serum 
the  growth  was  more  luxuriant  and  fluid  than  upon  the  solid  media. 
Upon  coagulated  serum  the  growth  was  somewhat  dry  and  elevated, 
and  was  frequently  so  loosely  attached  to  the  surface  of  the  medium 
as  to  be  readily  lifted  up  by  the  platinum  wire. 

The  growth  was  especially  luxuriant  upon  sheep's  blood-serum 
to  which  5  per  cent,  of  glycerin  was  added.  The  growth  upon  the 
Loffler  mixture  was  also  luxuriant. 

Upon  agar-agar  the  growth  was  more  meager;  it  was  more 
luxuriant  upon  glycerin  agar-agar  than  upon  plain  agar-agar,  the 
bacterial  mass  appearing  grayish  and  flatter  than  upon  blood- 
serum.  The  growth  never  extended  to  the  water  of  condensation 
to  form  a  floating  layer. 

The  bacillus  developed  well  upon  gelatin  after  it  had  grown  arti- 
ficially for  a  number  of  generations  and  become  accustomed  to  a 
saprophytic  existence.  Upon  the  surface  of  gelatin  the  growth  was 
in  general,  similar  to  that  upon  agar-agar.  In  puncture  cultures 
most  of  the  growth  occurred  upon  the  surface  to  form  a  whitish, 
grayish,  or  yellowish  wrinkled  layer.  Below  the  surface  of  the 
gelatin  the  growth  occurred  as  a  thick,  granular  column.  The 
medium  was  not  liquefied. 

In  bouillon,  growth  occurred  only  at  the  bottom  of  the  tube  in  the 
form  of  a  powdery  sediment. 


Cultivation  699 

Spronck*  believed  that  he  had  successfully  cultivated  the  organ- 
ism upon  glycerinized,  neutralized  potatoes,  first  seeing  the  growth 
after  the  lapse  of  ten  days.  Cultures  thus  prepared  were  found  to 
be  agglutinated  by  the  blood-serum  of  lepra  cases,  and  he  recom- 
mended the  agglutination  test  for  the  diagnosis  of  obscure  cases  of 
the  disease. 

Ducrey  claimed  to  have  cultivated  the  lepra  bacillus  in  grape- 
sugar,  agar,  and  in  bouillon  in  vacua.  His  results  need  confirmation. 

Rostf  claimed  to  have  isolated  and  cultivated  the  lepra  bacillus 
upon  media  free  from  sodium  chlorid.  The  technic  of  his  method 
is  thus  described  by  Rudolph :{ 

"  Small  lumps  of  pumice  stone  are  washed  and  then  dried  in  the  sun,  and  then 
allowed  to  absorb  a  mixture  of  i  ounce  of  meat  extract  and  2  ounces  of  water. 
This  pumice  stone  is  then  placed  in  wide-mouthed  bottles  and  placed  in  the  auto- 
clave. Each  bottle  is  provided  with  a  stopper  through  which  pass  two  tubes,  the 
one  tube  opening  into  the  autoclave  and  reaching  nearly  to  the  bottom  of  the 
bottle,  and  the  other  leading  from  the  top  of  the  bottle  into  a  condenser  adjoining. 
When  the  cover  of  the  autoclave  is  adjusted  and  the  steam  admitted,  then  in  the 
case  of  each  bottle,  the  steam  passes  by  the  one  tube  to  the  bottom  of  the  bottle, 
and  rising  through  the  pieces  of  pumice  stone,  the  steam,  carrying  with  it  the 
volatile  constituents  of  the  meat-extract,  reaches  the  condenser  by  the  second 
tube.  The  vapor  in  the  condenser  yields  the  salt-free  nutrient  medium  in  the 
proportion  of  2  liters  to  each  ounce  of  meat-extract  originally  used.  The  medium 
is  collected  from  the  condenser  in  sterilized  Pasteur  flasks  which  are  kept  plunged 
during  the  process  in  a  freezing  mixture  in  order  to  condense  some  of  the  volatile 
alkaloids  from  the  beef  that  would  otherwise  escape.  The  nutrient  fluid  is  now 
inoculated  with  the  bacillus  of  leprosy  and  the  flasks  kept  at  37°C.  for  from  four 
to  six  weeks;  at  the  end  of  this  period  when  examined  the  flasks  should  present  a 
turbid  appearance  with  a  stringy  white  deposit." 

Clegg§  announced  the  cultivation  of  lepra  bacilli  from  human 
leprous  tissue  in  symbiosis  with  ameba  and  other  bacteria.  The 
organisms  thus  cultured  he  kept  alive  in  subcultures.  The  method 
devised  by  Clegg  was  the  starting-point  of  a  more  extended  re- 
search by  Duval,||  who,  after  confirming  the  work  of  Clegg,  found 
that  the  bacillus  could  be  cultivated  directly  from  human  lesions 
upon  culture-media  containing  tryptophan,  without  the  symbiotic 
ameba  or  other  bacteria.  The  initial  culture  was  somewhat  difficult 
to  secure,  but  once  the  bacilli  grew,  transplantation  was  easily 
and  successfully  carried  on  for  indefinite  generations.  He  further 
found  that  the  lepra  bacillus  could  be  successfully  started  to  grow 
upon  the  ordinary  laboratory  media  if  bits  of  leprous  tissue  were 
placed  upon  them,  and  at  the  same  time  some  symbiotic  organism, 
such  as  the  colon,  typhoid,  proteus,  or  other  bacilli,  added.  Or 
if  the  tissue  were  already  contaminated  the  lepra  bacilli  proceeded 
to  multiply.  Duval  interprets  this  to  mean  that  the  lepra  bacillus 
is  unable  to  effect  the  destruction  of  the  albumin  molecule  alone,  and 

*  "Weekblad  van  het  Nederlandsch  Tijdschrift  voor  geneeskunde,"  Deel  n, 
1898,  No.  14;  abstract  "Centralbl.  f.  Bakt.,"  etc.,  1899,  xxv,  p.  257. 
t  "Brit.  Med.  Jour.,"  Feb.  22,  1905,  and  "Indian  Med.  Gazette,"  1905. 
t  "Medicine,", March,  1905,  p.  175. 
§  "Philippine  Journal  of  Science,"  1909,  rv,  403. 
1|  "Journal  of  Experimental  Medicine,"  1910,  xn,  649;  1911,  xm,  365. 


yoo  Leprosy 

hence  explains  the  advantage  of  adding  tryptophan.     The  medium 
most  successfully  employed  by  Duval  was  as  follows: 

"Egg-albumen  or  human  blood-serum  is  poured  into  sterile  Petri  dishes  and 
inspissated  for  three  hours  at  7o°C.  The  excised  leprous  nodule  is  then  cut  into 
thin  slices,  2  to  4  mm.  in  breadth  and  0.5  to  i  mm.  in  thickness,  which  are  dis- 
tributed over  the  surface  of  the  coagulated  albumin.  By  means  of  a  pipette  the 
medium  thus  seeded  with  bits  of  tissue  is  bathed  in  a  i  per  cent,  sterile  solution  of 
trypsin,  care  being  taken  not  to  submerge  the  pieces  of  leprous  tissue.  Sufficient 
fluid  is  added  to  moisten  thoroughly  the  surface  of  the  medium.  The  Petri  dishes 
are  now  placed  in  a  moist  chamber  at  37°C.,  and  allowed  to  incubate  for  a  week  or 
ten  days.  They  are  removed  from  the  plates  from  time  to  time,  as  evaporation 
necessitates,  for  the  addition  of  more  trypsin.  It  will  be  noted  that  after  a  week 
or  ten  days  the  tissue  bits  are  partially  sunken  below  the  surface  of  the  medium 
and  are  softened  to  a  thick,  creamy  consistence,  fragments  of  which  are  readily 
removed  with  a  platinum  needle.  On  microscopic  examination  of  this  material  it 
is  noted  that  the  leprosy  bacilli  have  increased  to  enormous  numbers  and  scarcely 
a  trace  of  the  tissue  remains.  Separate  lepra  bacillus  colonies  are  also  discernible 
on  and  around  the  softened  tissue  masses.  .  .  .  The  colonies  are  at  first  gray- 
ish white,  but  after  several  days  they  assume  a  distinct  orange-yellow  tint.  .  .  . 
Subcultures  may  be  obtained  by  transferring  portions  of  the  growth  to  a  second 
series  of  plates  or  to  slanted  culture-tubes  that  contain  the  special  albumin-trypsin 
medium.  After  the  third  or  fourth  generation  the  bacilli  may  be  grown  without 
difficulty  upon  glycerinated  serum  agar  prepared  in  the  following  manner: 

"Twenty  grams  of  agar,  3  gm.  of  sodium  chlorid,  30  cc.  of  glycerin,  and  500 
cc.  of  distilled  water  are  thoroughly  mixed,  clarified,  and  sterilized  in  the  usual 
way.  To  tubes  containing  10  cc.  of  this  material  is  added  in  proper  proportion  a 
solution  of  unheated  turtle  muscle  infusion.  Five  hundred  grams  of  turtle 
muscle  are  cut  into  fine  pieces  and  placed  in  a  flask  with  500  cc.  of  distilled  water. 
This  is  kept  in  the  ice-chest  for  forty-eight  hours  and  then  filtered  through  gauze 
to  remove  the  tissue.  The  filtrate  is  then  passed  through  a  Berkefeld  filter  for 
purposes  of  sterilization.  By  means  of  a  sterile  pipet,  5  cc.  of  the  muscle  filtrate 
is  added  to  the  agar  mixture  which  has  been  melted  and  cooled  to  42°C.  The 
tubes  are  now  thoroughly  agitated  and  allowed  to  solidify  in  the  slanted  position. 

"This  medium  is  perfectly  clear  or  of  a  light  amber  color,  and  admirably  suited 
to  the  cultivation  of  the  Bacillus  lepra,  once  the  initial  culture  has  been  started. 
Growth  is  luxuriant  and  reaches  its  maximum  in  forty-eight  to  sixty  hours.  On 
the  surface  of  this  medium  the  growth  is  moi«t  and  orange-yellow  in  color,  while 
in  the  water  of  condensation,  though  growth  apparently  has  not  occurred,  the 
detached  bacilli  collect  in  the  dependent  parts  in  the  form  of  feathery  masses 
without  clouding  the  fluid. 

"  Ordinary  nutrient  agar  may  be  used  with  trypsin  as  a  plating  medium  instead 
of  the  inspissated  serum  where  bits  of  tissue  are  employed.  With  the  addition  of 
i  per  cent,  of  tryptophan  it  answers  every  purpose,  whether  the  bacilli  are  planted 
with  tissue  or  alone.  It  also  serves  to  start  multiplication  of  lepra  bacilli  that 
are  contaminated  at  the  time  of  plating.  In  the  latter  case  the  medium  is 
'surface  seeded'  with  an  emulsion  of  the  tissue  juices  in  the  same  manner  as  in 
preparing  '  streak '  plates.  The  leprosy  colonies  in  the  thinner  parts  of  the  loop 
track  are  well  separated  and  easily  distinguished  from  those  of  other  species  by 
their  color  and  by  their  appearance  only  after  two  to  five  days. 

"In  using  an  agar  medium  it  is  well  to  leave  out  the  peptone  and  to  titrate  the 
reaction  to  1.5  per  cent,  alkaline  in  order  to  prevent  too  profuse  growth  of  the 
associated  bacteria;  besides,  an  alkaline  medium  seems  best  adapted  for  the 
multiplication  of  the  lepra  bacillus. 

"Bacillus  leprae  will  also  grow  on  the  various  blood-agar  media  once  they  are 
accustomed  to  artificial  conditions.  The  Novy-McNeal  agar  for  the  cultivation 
of  trypanosomes  gives  a  luxuriant  growth  of  the  organism  if  2  per  cent,  glycerin 
has  been  added;  without  the  glycerin,  growth  is  very  scant.  Fluid  media  are 
not  suited  for  the  artificial  cultivation  of  leprosy  bacilli  unless  they  are  kept  upon 
the  surface.  Like  the  tubercle  bacilli  they  require  abundant  oxygen.  .  .  . 

"Ordinarily  the  growth  of  Bacillus  leprae  is  very  moist,  and  in  this  respect 
unlike  that  of  Bacillus  tuberculosis,  except  possibly  the  avian  stain.  Sometimes 
when  the  medium  is  devoid  of  water  of  condensation,  the  growth  is  dry  and  occa- 
sionally wrinkled,  though  it  is  easily  removed  from  the  surface  of  the  medium 


Pathogenesis  701 

"The  chromogenic  property  of  lepra  cultures  is  a  constant  and  characteristic 
feature  of  the  rapidly  growing  strains.  The  color  varies  in  the  degree  of  intensity 
depending  upon  the  medium  employed.  If  glycerinated  agar  (without  peptone) 
is  used,  the  colonies  are  faint  lemon,  while  on  inspissated  blood-serum  they  are 
deep  orange.  It  is  noteworthy  that  the  growth  in  the  tissues  and  in  the  first 
dozen  or  so  generations  on  artificial  media  is  entirely  without  pigment." 

Although  each  of  the  workers  upon  leprosy  has  begun  by  asserting 
that  he  had  certainly  cultivated  the  specific  organism,  a  time  comes 
when  a  more  extended  acquaintance  with  the  bacteriology  of  the 
disease  seems  to  cause  him  to  doubt  the  results  of  his  own  work. 
This  is  particularly  true  of  this  work  of  Duval,  which  was  prosecuted 
with  enthusiasm,  carried  conviction  with  it,  and  then  was  partially 
repudiated  by  its  author,  for  in  the  discussion  before  the  iyth  Inter- 
national Medical  Congress  in  London  in  1913,  Duval*  is  reported  as 
saying  that  "he  knew  less  of  the  bacteriology  of  leprosy  now  than  he 
did  some  four  years  ago.  He  had  made  several  mistakes,  had 
stated  openly  that  he  had  cultivated  the  leprosy  bacillus,  but  now 
admitted  frankly  that  he  was  mistaken." 

The  interesting  question  that  awaits  settlement  now  seems  to  be,  if 
these  bacilli,  and  specially  the  bacillus  of  Duval,  are  not  Bacillus 
leprae,  what  are  they?  What  relation  do  they  bear  to  leprosy? 

Pathogenesis. — Melcher  and  Ortmann*  introduced  fragments 
of  lepra  nodules  into  the  anterior  chambers  of  the  eyes  of  rabbits, 
and  observed  the  death  of  the  animals  after  some  months,  with  what 
they  considered  to  be  typical  leprous  lesions  of  all  the  viscera, 
especially  the  cecum;  but  the  later  careful  experiments  of  Tashirof 
show  that  most  of  the  lower  animals  are  entirely  insusceptible  to 
infection  with  the  lepra  bacillus,  and  that  when  they  are  inoculated 
the  bacilli  persistently  diminish  in  numbers  and  finally  disappear. 

NicolleJ  found  it  possible  to  infect  monkeys  with  material  rich 
in  lepra  bacilli  taken  from  human  beings.  The  lesions  appeared 
only  after  an  incubation  period  that  was  in  some  cases  prolonged 
from  twenty-two  to  ninety-four  days.  The  lesions  persisted  but  a 
short  time  and  the  monkeys  recovered  in  from  thirty  to  one  hundred 
and  fifty  days. 

Clegg§  and  Sugai||  found  Japanese  dancing  mice  susceptible 
to  infection  with  leprous  material,  the  micro-organisms  not  remain- 
ing localized  at  the  seat  of  inoculation,  but  disseminating  through- 
out the  animal's  body.  Their  observation  has  been  confirmed  by 
Duval,**  who  laterft  was  also  able  to  infect  monkeys — Macacus 
rhesus — -with  pure  cultures  of  the  organism  and  produce  the  typical 
disease. 

*  "Berliner  klin.  Wochenschrift,"  1885-1886. 

t  "Centralbl.  f.  Bakt.  u.  Parasitenk,"  (Originale),  March  12,  i9O2,xxxi,No. 
7,  p.  276. 

J  "Semaine  medicale,"  1905,  No.  10,  p.  no. 

§  "Philippine  Journal  of  Science,"  1909,  rv,  403. 

|]  "Lepra,"  1909,  vm,  203. 

**  "Journal  of  Experimental  Medicine,"  1910,  xn,  649. 
tflbid.,  191 1,  xm,  374. 


702  Leprosy 

Very  few  instances  are  recorded  in  which  actual  inoculation  has 
produced  leprosy  in  man.  Arning*  was  able  to  experiment  upon  a 
condemned  criminal,  of  a  family  entirely  free  from  the  disease,  in 
the  Sandwich  Islands.  Fragments  of  tissue  freshly  excised  from  a 
lepra  nodule  were  introduced  beneath  his  skin  and  the  man  was 
kept  under  observation.  In  the  course  of  some  months  typical 
lesions  began  to  develop  at  the  points  of  inoculation  and  spread 
gradually,  ending  in  general  leprosy  in  about  five  years. 

S ticker  f  is  of  the  opinion  that  the  primary  infection  in  lepra 
takes  place  through  the  nose,  supporting  his  opinion  by  observa- 
tions upon  153  accurately  studied  cases,  in  which — • 

1.  The  nasal  lesion  is  the  only  one  constant  in  both  the  nodular 
and  anesthetic  forms  of  the  disease. 

2.  The  nasal  lesion  is  peculiar — -i.e.,  characteristic — -and  entirely 
different  from  all  other  lepra  lesions. 

3.  The  clinical  symptoms  of  lepra  begin  in  the  nose. 

4.  The  relapses  in  the  disease  always  begin  with  nasal  symptoms, 
such  as  epistaxis,  congestion  of  the  nasal  mucous  membrane,   a 
sensation  of  heat,  etc. 

5.  In  incipient  cases  the  lepra  bacilli  are  first  found  in  the  nose. 
Lesions. — -The    lepra    nodes    in    general    resemble    tuberculous 

lesions,  but  are  superficial,  affecting  the  skin  and  subcutaneous 
tissues.  Rarely  they  may  also  occur  in  the  organs.  VirchowJ 
has  seen  a  case  in  which  lepra  bacilli  could  be  found  only  in  the 
spleen. 

Once  established  in  the  body,  the  bacillus  may  grow  in  the  con- 
nective tissues  and  produce  chronic  inflammatory  nodes — 'the 
analogues  of  tubercles; — or  in  the* nerves,  causing  anesthesia  and 
trophic  disturbances.  On  this  account  two  forms  of  the  disease, 
lepra  nodosa  (elephantiasis  graecorum)  and  lepra  anczsthetica,  are 
described.  These  forms  may  occur  independently  of  one  another, 
or  may  be  associated  in  the  same  case. 

The  nodes  consist  of  lymphoid  and  epithelioid  cells  and  fibers, 
and  are  vascular,  so  that  much  of  the  embryonal  tissue  completes 
its  transformation  to  fibers  without  necrotic  changes.  This  makes 
the  disease  productive  rather  than  destructive,  the  lesions  re- 
sembling new  growths.  The  bacilli,  which  occur  in  enormous 
numbers,  are  often  found  in  groups  inclosed  within  the  protoplasm 
of  certain  large  vacuolated  cells — 'the  "lepra  cells" — -which  seem  to 
be  partly  degenerated  endothelial  cells.  Sometimes  they  are 
anuclear;  rarely  they  contain  several  nuclei  (giant  cells).  Bacilli 
also  occur  in  the  lymph-spaces  and  in  the  nerve-sheath. 

Lepra  nodules  do  not  degenerate  like  tubercles,  and  the  ulcera- 
tion,  which  constitutes  a  large  part  of  the  pathology  of  the  disease, 

*  "Centralbl.  f.  Bakt.,"  etc.,  1889,  vi,  p.  201. 

f  "  Mittheilungen  und  Verhandlungen  der  internationalen  wissenschaf  tlichen 
Lepra- Konferenz  zu  Berlin,"  Oct.,  1897,  2,  Theil. 
J  Ibid. 


Lesions 


703 


seems  to  be  largely  due  to  the  injurious  action  of  external  agencies 
upon  the  feebly  vital  pathologic  tissue. 

According  to  the  studies  of  Johnston  and  Jamieson,*  the  bacterio- 
logic  diagnosis  of  nodular  leprosy  can  be  made  by  spreading  serum 
obtained  by  scraping  a  leprous  nodule  upon  a  cover-glass,  drying, 
fixing,  and  staining  with  carbol-fuchsin  and  Gabbet's  solution  as 
for  the  tubercle  bacillus.  In  such  preparations  the  bacilli  are  pres- 
ent in  enormous  numbers,  forming  a  marked  contrast  to  tuber- 
culous skin  diseases,  in  which  they  are  very  few. 


Fig.  284. — Lepra  anaesthetic  a  (McConnell). 

In  anesthetic  leprosy  nodules  form  upon  the  peripheral  nerves, 
and  by  connective-tissue  formation,  as  well  as  by  the  entrance  of 
the  bacilli  into  the  nerve-sheaths,  cause  irritation,  followed  by 
degeneration  of  the  nerves.  The  anesthesia  following  the  peripheral 
nervous  lesions  predisposes  to  the  formation  of  ulcers,  etc.,  by  allow- 
ing injuries  to  occur  without  detection  and  to  progress  without 
observation.  The  ulcerations  of  the  hands  and  feet,  with  frequent 
loss  of  fingers  and  toes,  follow  these  lesions,  probably  in  the  same 
manner  as  in  syringomyelia. 

The  disease  usually  first  manifests  itself  upon  the  face,  extensor 
surfaces,  elbows,  and  knees,  and  for  a  long  time  confines  itself  to 
*  "Montreal  Med.  Journal,"  Jan.,  1897. 


704 


Leprosy 


the  skin.  Ultimately  it  sometimes  invades  the  lymphatics  and  ex- 
tends to  the  internal  viscera.  Death  ultimately  occurs  from  ex- 
haustion, if  not  from  the  frequent  intercurrent  affections,  especially 
pneumonia  and  tuberculosis,  to  which  the  patients  seem  predisposed. 

Specific  Therapy. — Carrasquilla's*  "leprosy  serum"  was  prepared 
by  injecting  the  serum  separated  from  blood  withdrawn  from 
lepers,  into  horses,  mules,  and  asses,  and,  after  a  number  of  in- 
jections, bleeding  the  animals  and  separating  the  serum.  There  is 
no  reason  for  thinking  that  such  a  product  could  have  therapeutic 
value.  In  practice  it  proved  worthless. 

Rostf  prepared  massive  cultures  of  the  lepra  bacillus,  filtered 


Fig.  285. — A  case  of  lepra  nodosa  treated  in  the  Medico-Chirurgical  Hospital  of 

Philadelphia. 

them  through  porcelain,  concentrated  the  nitrate  to  one-tenth  of  its 
volume,  and  mixed  the  filtrate  with  an  equal  volume  of  glycerin. 
The  resulting  preparation  was  called  leprolin  and  was  supposed  to  be 
analogous  to  tuberculin.  With  it  he  treated  a  number  of  lepers 
at  the  Leper  Hospital  at  Rangoon,  Burmah,  many  of  whom  greatly 
improved  and  some  of  whom  seemed  to  be  cured.  Confirmation  of 
the  work  by  others  is  greatly  desired. 
Sanitation. — While  not  so  contagious  as  tuberculosis,  it  has 

*  "Wiener  med.  Wochenschrift,"  No.  41,  1897. 
t  "Brit.  Med.  Jour.,"  Feb.  n,  1905. 


Sanitation  705 

been  proved  that  leprosy  is  transmissible,  and  it  may  be  regarded 
as  an  essential  sanitary  precaution  that  lepers  should  be  segregated 
and  mingle  as  little  as  possible  with  healthy  persons.  The  disease 
is  not  hereditary,  so  that  there  is  no  reason  why  lepers  should  not 
marry  among  themselves.  The  children  should,  however,  be  taken 
from  the  parents  lest  they  be  subsequently  infected. 

45 


CHAPTER  XXXI 

GLANDERS 

BACILLUS  MALLEI  (LOFFLER  AND  SCHUTZ)* 

General  Characteristics. — A  non-motile,  non-flagellate,  non-sporogenous,  non- 
liquefying,  non-chromogenic,  non-aerogenic,  aerobic  and  optionally  anaerobic, 
acid-forming  and  milk  coagulating  bacillus,  pathogenic  for  man  and  the  lower 
animals,  staining  by  ordinary  methods,  but  not  by  Gram's  method. 

Glanders,  "Rotz"  (German)  OT-"morve"  (French),  is  an  infectious 
mycotic  disease  which,  fortunately,  is  almost  entirely  confined  to 
the  lower  animals.  Only  occasionally  does  it  secure  a  victim  among 
hostlers,  drovers,  soldiers,  and  others  whose  vocations  bring  them  in 
contact  with  diseased  horses.  Several  bacteriologists  have  succumbed 
to  accidental  laboratory  infection. 

Glanders  was  first  known  to  us  as  a  disease  of  the  horse  and 
ass,  characterized  by  the  formation  of  discrete,  cleanly  cut  ulcers 
upon  the  mucous  membrane  of  the  nose.  The  ulcers  in  the  nose 
are  formed  by  the  breaking  down  of  inflammatory  nodules  which 
can  be  detected  in  all  stages  upon  the  diseased  membranes.  Hav- 
ing once  formed,  they  show  no  tendency  to  recover,  but  slowly 
spread  and  persistently  discharge  a  virulent  pus.  The  edges  of 
the  ulcers  are  indurated  and  elevated,  their  surfaces  often  smooth. 
The  disease  does  not  progress  to  any  great  extent  before  the  sub- 
maxillary  lymphatic  glands  begin  to  enlarge,  soften,  and  ulcerate. 
The  lungs  may  also  become  infected  by.  inspiration  of  the  infectious 
material  from  the  nose  and  throat,  and  contain  small  foci  of  broncho- 
pneumonia  not  unlike  tubercles  in  their  early  appearance.  The 
animals  ultimately  die  of  exhaustion. 

Specific  Organism. — In  1882,  shortly  after  the  discovery  of  the 
tubercle  bacillus,  Loftier  and  Schiitz  discovered  in  the  discharges 
and  tissues  of  the  disease  the  specific  micro-organism,  the  glanders 
bacillus  (Bacillus  mallei). 

Distribution. — The  glanders  bacillus  does  not  seem  to  find  con- 
ditions outside  the  animal  body  suitable  for  its  growth,  and  prob- 
ably lives  a  purely  parasitic  existence. 

Morphology. — 'The  glanders  bacillus  is  somewhat  shorter  and 
distinctly  thicker  than  the  tubercle  bacillus,  and  has  rounded  ends. 
It  measures  about  0.25  to  0.4  X  1.5  to  3  /*,  and  is  slightly  bent. 
Coccdid  and  branched  forms  sometimes  occur.  It  usually  occurs 
singly,  though  upon  blood-serum,  and  especially  upon  potato, 

*  "Deutsche  med.  Wochenschrift,"  1882,  52. 
706 


Staining 


707 


conjoined  individuals  may  occasionally  be  found.  Long  threads 
are  never  formed. 

When  stained  with  ordinary  aqueous  solutions  of  the  aniline 
dyes,  or  with  Loffler's  alkaline  methylene-blue,  the  bacillary  sub- 
stance does  not  usually  appear  homogeneous,  but,  like  that  of  the 
diphtheria  bacillus,  shows  marked  inequalities,  some  areas  being 
deeply,  some  faintly,  stained. 

The  bacillus  is  non-motile,  has  no  flagella,  and  does  not  form 
spores. 

Staining.  —  'The  organism  can  be  stained  with  the  watery  anilin- 
dye  solutions,  but  not  by  Gram's  method.  The  bacillus  readily 
gives  up  the  stain  in  the  presence  of  decolorizing  agents,  so  is  dif- 


^  i  g^fy-w  J&     V 

:>  xLi:^x          *1*  r  H 


Fig.   286. — Bacillus  mallei,  from  a  culture  upon  glycerin  agar-agar.      X    1000 
(Frankel  and  PfeifTer). 

ficult  to  stain  in  tissues.  Lofner  accomplished  the  staining  by 
allowing  the  sections  to  lie  for  some  time  (five  minutes)  in  the  alka- 
line methylene-blue  solution,  then  transferring  them  to  a  solution  of 
sulphuric  and  oxalic  acids: 

Concentrated  sulphuric  acid 2  drops 

Five  per  cent,  oxalic  acid  solution i  drop 

Distilled  water 10  cc. 

for  five  seconds,  then  to  absolute  alcohol,  xylol,  etc.  The  bacilli 
appear  dark  blue  upon  a  paler  ground.  This  method  gives  very  good 
results,  but  has  been  largely  superseded  by  the  use  of  Kiihne's  car- 
bolmethylene-blue. 

Methylene-blue i .  5 

Alcohol 10 .  o 

Five  per  cent,  aqueous  phenol  solution 100 .  o 

Kiihne  stains  the  section  for  about  half  an  hour,  washes  it  in  water, 


708  Glanders 

decolorizes  it  carefully  in  hydrochloric  acid  (10  drops  to  500  cc.  of 
water),  immerses  it  at  once  in  a  solution  of  lithium  carbonate  (8  drops 
of  a  saturated  solution  of  lithium  carbonate  in  10  cc.  of  water), 
places  it  in  a  bath  of  distilled  water  for  a  few  minutes,  dips  it  into 
absolute  alcohol  colored  with  a  little  methylene-blue,  dehydrates  it 
in  anilin  oil  containing  a  little  methylene-blue  in  solution,  washes  it 
in  pure  anilin  oil,  not  colored,  then  in  a  light  ethereal  oil,  clears  it  in 
xylol,  and  finally  mounts  it  in  balsam. 

Vital  Resistance. — The  organism  grows  only  between  25°  and  42 °C. 
It  is  killed  by  exposure  to  60° C.  for  two  hours,  or  to  75°C.  for  one 
hour.  Sunlight  kills  it  after  twenty-four  hours'  exposure.  Thor- 
ough drying  destroys  it  in  a  short  time.  When  planted  upon  cul- 
ture-media, sealed,  and  kept  cool  and  in  the  dark,  it  may  be  kept 
alive  for  months  and  even  years.  Exposure  to  i  per  cent,  carbolic 
acid  destroys  it  in  about  half  an  hour;  i  :  1000  bichlorid  of 
mercury  solution,  in  about  fifteen  minutes.  According  to  Hiss 
and  Zinsser,  it  may  remain  alive  in  the  water  of  horse-troughs  for 
seventy  days. 

Isolation. — Attempts  to  isolate  the  glanders  bacillus  from  infectious 
discharges,  by  the  usual  plate  method,  are  apt  to  fail,  on  account 
of  the  presence  of  other  more  rapidly  growing  organisms. 

A  better  method  seems  to  be  by  infecting  an  animal  and  recover- 
ing the  bacillus  from  its  tissues.  For  this  purpose  the  guinea-pig, 
being  a  highly  susceptible  as  well  as  a  readily  procurable  animal,  is 
appropriate.  When  a  subcutaneous  inoculation  of  some  of  the 
infectious  pus  is  made,  a  tumefaction  can  be  observed  in  guinea- 
pigs  in  from  four  to  five  days.  Somewhat  later  this  tumefac- 
tion changes  to  a  caseous  nodule,  which  ruptures  and  leaves  a 
chronic  superficial  ulcer  with  irregular  margins.  The  lymph- 
glands  speedily  become  invaded,  and  in  four  or  five  weeks  signs 
of  general  infection  appear. .  The  lymph-glands,  especially  of  the 
inguinal  region,  suppurate,  and  the  testicles  frequently  undergo 
the  same  process.  Later  the  joints  are  affected  with  a  suppura- 
tive  arthritis,  the  pus  from  which  contains  the  bacilli.  The 
animal  eventually  dies  of  exhaustion.  No  nasal  ulcers  form  in 
guinea-pigs. 

In  field-mice  the  disease  is  much  more  rapid,  no  local  lesions 
being  visible.  For  two  or  three  days  the  animal  seems  unwell,  its 
breathing  is  hurried,  it  sits  with  closed  eyes  in  a  corner  of  the  cage, 
and  finally,  without  any  other  preliminaries,  tumbles  over  dead. 

From  the  tissues  of  the  inoculated  animals  pure  cultures  are  easily 
made.  Perhaps  the  best  places  from  which  to  secure  a  culture  are 
the  softened  nodes  which  have  not  ruptured,  or  the  joints. 

Diagnosis  of  Glanders. — Straus*  has  given  us  a  method  which 
is  of  great  use,  both  for  isolating  pure  cultures  of  the  glanders  bacillus 
and  for  making  a  diagnosis  of  the  disease. 

*  "  Compt.  rendu  Acad.  d.  Sciences,"  Paris,  cvm,  530. 


Cultivation  709 

But  a  short  time  is  required.  The  material  suspected  to  contain  the  glanders 
bacillus  is  injected  into  the  peritoneal  cavity  of  a  male  guinea-pig.  In  three  or 
four  days  the  disease  becomes  established  and  the  testicles  enlarge;  the  skin  over 
them  becomes  red  and  shining;  the  testicles  themselves  begin  to  suppurate,  and 
often  evacuate  through  the  skin.  The  animal  dies  in  about  two  weeks.  If, 
however,  it  be  killed  and  its  testicles  examined,  the  tunica  vaginalis  testis  will  be 
found  to  contain  pus,  and  sometimes  to  be  partially  obliterated  by  inflammatory 
exudation.  The  bacilli  are  present  in  this  pus,  and  can  be  secured  from  it  in  pure 
cultures. 

The  value  of  Straus'  method  has  been  somewhat  lessened  by  the  dis- 
covery by  Kutcher,*  that  a  new  bacillus,  which  he  has  classed  among 
the  pseudo- tubercle  bacilli,  produces  a  similar  testicular  swelling  when 
injected  into  the  abdominal  cavity ;  also  by  Levy  and  Steinmetz,  f  who 
found  that  Staphylococcus  pyogenes  aureus  was  also  capable  of  pro- 
voking suppurative  orchids.  However,  the  diagnosis  is  certain  if  a 
culture  of  the  glanders  bacillus  be  secured  from  the  pus  in  the  scrotum. 

For  the  diagnosis  of  the  disease  in  living  animals,  subcutaneous 
injections  of  mallein  (q.v.}  are  also  employed. 

McFadyenJ  was  the  first  to  recommend  agglutination  of  the 
glanders  bacillus  by  the  serum  of  supposedly  infected  animals  as  a 
test  of  the  existence  of  glanders.  The  subject  has  been  somewhat 
extensively  tried  and  officially  adopted  by  the  Prussian  govern- 
ment. Moore  and  Taylor,  §  in  a  recent  review  and  examination  of 
the  test,  conclude  that  it  is  easier  and  quite  as  accurate  as  the  mallein 
method  and  is  applicable  in  cases  where  fever  exists.  The  maximum 
dilution  of  normal  horse-serum  that  will  macroscopically  agglutinate 
glanders  bacilli  is  i  :  500,  but  occurs  in  very  few  cases.  The  maxi- 
mum agglutinative  power  of  the  serum  of  diseased  horses  not  suffer- 
ing from  glanders  is  not  higher  than  that  of  normal  serum.  The 
diagnosis  is  usually  not  difficult  to  make,  but  requires  much  care. 
Cultures  of  the  glanders  bacillus  sometimes  unexpectedly  lose  their 
ability  to  agglutinate. 

The  diagnosis  of  glanders  by  means  of  the  complement-fixation 
method  has  been  tried  with  glittering  results  by  Mohler  and  Eichhorn.  || 

Cultivation. — The  bacillus  is  an  aerobic  and  optionally  anaerobic 
organism,  and  can  be  grown  in  bouillon,  upon  agar-agar,  better  upon 
glycerin  agar-agar,  very  well  upon  blood-serum,  and  quite  character- 
istically upon  potato.  The  optimum  temperature  is  37.5°C. 

Colonies. — Upon  4  per  cent,  glycerin  agar-agar  plates  the  colonies 
appear  upon  the  second  day  as  whitish  or  pale  yellow,  shining,  round 
dots.  Under  the  microscope  they  are  brownish  yellow,  thick  and 
granular,  with  sharp  borders. 

Bouillon. — In  broth  cultures  the  glanders  bacillus  causes  turbidity, 
the  surface  of  the  culture  being  covered  by  a  slimy  scum.  The 
medium  becomes  brown  in  color. 

*  "Zeitschrift  fur  Hygiene,"  Bd.  xxi,  Heft  i,  Dec.  6,  1895. 
f  "Berliner  klin.  Wochenschrift,"  March  18,  1895,  No.  n. 
t  "Jour.  Comp.  Path,  and  Therap.,"   1896,  p.  322. 
§  "Jour.  Infectious  Diseases,"  1907,  rv,  p.  85,  supplement. 
||  "Report  of  the  Bureau  of  Animal  Industry,"  1910. 


710 


Glanders 


Gelatin  is  not  liquefied.  The  growth  upon  the  surface  is  grayish 
white  and  slimy,  never  abundant. 

Agar-agar. — 'Upon  agar-agar  and  glycerin  agar-agar  the  growth 
occurs  as  a  moist  shining  viscid  layer. 

Blood-serum. — Upon  blood-serum  the  growth  is  rather  character- 
istic, the  colonies  along  the  line  of  inoculation  appearing  as  cir- 
cumscribed, clear,  transparent  drops,  which  later  become  confluent 
and  form  a  transparent  layer  unaccompanied  by  liquefaction. 

Potato. — The  most  characteristic  growth  is  upon  potato.  It  first 
appears  in  about  forty-eight  hours  as  a  transparent,  honey-like, 
yellowish  layer,  developing  only  at  incubation  temperatures,  and 
soon  becoming  reddish-brown  in  color.  As  this  brown  color  of  the 
colony  develops,  the  potato  for  a  considerable  distance  around  it 
becomes  greenish  brown.  Bacillus  pyocyaneus  sometimes  produces 
somewhat  the  same  appearance. 


Fig.  287. — Culture  of  glanders  upon  cooked  potato  (Loffler). 

Milk. — In  litmus  milk  the  glanders  bacillus  produces  acid.  A 
firm  coagulum  forms  and  subsequently  separates  from  the  clear 
reddish  whey. 

Metabolic  Products. — The  organism  produces  acids  and  curdling 
ferments.  It  forms  no  indol,  no  liquefying  or  proteolytic  ferments. 
There  is  no  exotoxin.  All  the  poisonous  substances  seem  to  be 
endotoxins. 

Mallein. — Babes,*  Bonome,f  Pearson, {  and  others  have  prepared 
a  substance,  mallein,  from  cultures  of  the  glanders  bacillus,  and  have 
employed  it  for  diagnostic  purposes.  It  seems  to  be  useful  in  veteri- 

*  "Archiv  de  Med.  exp.  et  d'Anat.  patholog.,"  1892,  No.  4. 

"Deutsche  med.  Woch.,"  1894,  Nos.  36  and  38,  pp.  703,  725,  and  744. 
j  "Jour,  of  Comp.  Med.  and  Vet.  Archiv,"  Phila.,  1891,  xn,  pp.  411-415. 


Lesions  711 

nary  medicine,  the  reaction  following  its  injection  into  glandered 
animals  being  similar  to  that  caused  by  the  injection  of  tuberculin 
into  tuberculous  animals.  The  preparation  of  mallein  is  simple. 
Cultures  of  the  glanders  bacillus  are  grown  in  glycerin  bouillon  for 
several  weeks  and  killed  by  heat.  The  culture  is  then  filtered 
through  porcelain,  to  remove  the  dead  bacteria,  and  evaporated  to 
one-tenth  of  its  volume.  Before  use  the  mallein  is  diluted  with 
nine  times  its  volume  of  0.5  per  cent,  aqueous  carbolic  acid  solution. 
The  dose  for  diagnostic  purposes  is  0.25  cc.  for  the  horse.  It  has 
also  been  prepared  from  potato  cultures,  which  are  said  to  yield 
a  stronger  product.  The  agent  is  employed  exactly  like  tuberculin, 
the  temperature  being  taken  before  and  after  its  hypodermic  in- 
jection. A  febrile  reaction  of  more  than  i .  5°C.  is  said  to  be  indicative 
of  the  disease. 

Pathogenesis. — That  the  bacillus  is  the  cause  of  glanders  there  is 
no  room  to  doubt,  as  Lofner  and  Schiitz  have  succeeded,  by  the 
inoculation  of  horses  and  asses,  in  producing  the  well-known  disease. 

The  goat,  cat,  hog,  field-mouse,  wood-mouse,  marmot,  rabbit, 
guinea-pig,  and  hedgehog  all  appear  to  be  susceptible.  Cattle, 
house-mice,  white  mice,  rats,  and  birds  are  immune. 

Infection  may  take  place  through  the  mucous  membranes  of  the 
nose,  mouth,  or  alimentary  tract,  and  apparently  without  preexisting 
demonstrable  lesions. 

The  disease  assumes  either  an  acute  form,  characterized  by  de- 
structive necrosis  and  ulceration  of  the  mucous  membranes  with 
fever  and  prostration,  terminating  in  pneumonia,  or,  as  is  more 
frequent,  a  chronic  form  ("farcy"),  in  which  the  lesions  of  the 
mucous  membranes  are  less  destructive  and  in  which  there  is  a 
generalized  distribution  of  the  micro-organisms  throughout  the  body, 
with  resulting  more  or  less  widespread  nodular  formations  (farcy- 
buds)  in  the  skin.  The  acute  form  is  quickly  fatal,  death  some- 
times coming  on  in  from  four  to  six  weeks;  the  chronic  form  may  last 
for  several  years  and  end  in  complete  recovery. 

Lesions. —  When  stained  in  sections  of  tissue  the  bacilli  are  found 
in  small  inflammatory  areas.  These  nodules  can  be  seen  with  the 
naked  eye  scattered  through  the  liver,  kidney,  and  spleen  of  animals 
dead  of  experimental  glanders.  They  consist  principally  of  leuko- 
cytes, but  also  contain  numerous  epithelioid  cells.  As  is  the  case 
with  tubercles,  the  centers  of  the  nodules  are  prone  to  necrotic 
changes,  but  the  cells  show  marked  karyorrhexis,  and  the  tendency 
is  more  toward  colliquation  than  caseation.  The  typical  ulcerations 
depend  upon  retrogressive  changes  occurring  upon  mucous  surfaces, 
the  breaking  down  of  the  nodules  permitting  the  softened  material 
to  escape.  At  times  the  lesions  heal  with  the  formation  of  stellate 
scars. 

Baumgarten*  regarded  the  histologic  lesions  of  glanders  as  much 

*  "  Pathologische  Mykologie,"  Braunschweig,  1890. 


712 


Glanders 


Fig.  288. — Pustular  eruption  of  acute  glanders  as  exhibited  on  the  day  of  the 
patient's  death,  twenty-eight  days  after  initial  chill  (Zeit). 


Fig.  289. — Lesions  of  glanders  in  the  skin  of  a  horse.  (Kitt). 


Glanders  in  Human  Beings  713 

like  those  of  the  tubercle.  He  first  saw  epithelioid  cells  accumulate, 
followed  by  the  invasion  of  leukocytes.  Tedeschi*  was  not  able  to 
confirm  Baumgarten's  work,  but  found  the  primary  change  to  be 
necrosis  of  the  affected  tissue  followed  by  invasion  of  leukocytes. 
The  observations  of  Wrightf  are  in  accord  with  those  of  Tede- 
schi. He  first  saw  a  marked  degeneration  of  the  tissue,  and  then 
an  inflammatory  exudation,  amounting  in  some  cases  to  actual 
suppuration. 

Glanders  in  Human  Beings. — Human  beings  are  but  rarely  in- 
fected.    The  disease  has,  however,  occurred  among  those  in  frequent 


Fig.  290. — Farcy  affecting  the  skin  of  the  shoulder  (Mohler  and  Eichhorn,  in 
Twenty-seventh  Annual  Report  of  the  Bureau  of  Animal  Industry,  U.  S.  De- 
partment of  Agriculture,  1910). 

contact  with  horses  and  among  bacteriologists.  It  occurs  either 
in  an  acute  form  in  which,  from  whatever  primary  focus  may  have 
been  its  starting-point,  the  distribution  of  micro-organisms  may 
be  so  rapid  as  to  induce  an  affection  with  skin  lesions'  resembling 
smallpox  and  terminating  fatally  in  eight  or  ten  days. 

The  chronic  form  in  man  is  chiefly  confined  to  the  nasal  and  laryn- 
geal  mucosa.  It  is  commonly  mistaken  for  more  simple  infections, 
and  though  it  sometimes  shows  its  character  by  generalizing,  it  not 
infrequently  recovers. 

Virulence. — The  organism  is  said  to  lose  virulence  if  cultivated 
for  many  generations  upon  artificial  media.  While  this  is  true, 
attempts  to  attenuate  fresh  cultures  by  heat,  etc.,  have  usually 
failed. 

*  "Zeigler's  Beitrage  z.  path.  Anat.,"  Bd.  xin,  1893. 

f  "  Journal  of  Experimental  Medicine,"  vol.  i,  No.  4,  p.  577. 


Glanders 


Immunity. — Leo  has  pointed  out  that  white  rats,  which  are  im- 
mune to  the  disease,  may  be  made  susceptible  by  feeding  with 
phloridzin  and  causing  glycosuria. 

Babes  has  asserted  that  the  injection  of  mallein  into  susceptible 
animals  will  immunize  them  against  glanders.  Some  observers 
claim  to  have  seen  good  therapeutic  results  follow  the  repeated  in- 


Fig.  291. — Lesions  of  glanders  in  the  nasal  septum  of  a  horse  (Mohler  and 
Eichhorn,  in  Twenty-seventh  Annual  Report  of  the  Bureau  of  Animal  Industry, 
U.  S.  Department  of  Agriculture,  1910). 

jection  of  mallein  in  small  doses.  Others,  as  Chenot  and  Picq,* 
find  blood-serum  from  immune  animals  like  the  ox  to  be  curative 
when  injected  into  guinea-pigs  infected  with  glanders. 

Pseudo -glanders  Bacillus. — 'Bacilli  similar  to  the  glanders  bacillus 
in  tinctorial  and  cultural  peculiarities,  but  not  pathogenic  for  mice, 
guinea-pigs,  or  rabbits,  have  been  isolated  by  Babes,  f  and  by  Selter,t 
and  called  the  pseudo-glanders  bacillus. 


*  " Compte-rendu  de  la  Soc.  de  Biol.,"  March  26,  1892. 

f  "Archiv  de  med.  exp.  et  d'anat.  path.,"  1891. 

j  "Centralbl.  f.  Bakt.,"  etc.,  Feb.  18,  1902,  xxxv,  5,  p. 


529- 


CHAPTER  XXXII 
RHINOSCLEROMA 

BACILLUS  RHINOSCLEROMATIS  (VON  FRISCH*) 

General  Characteristics. — A  non-motile,  non-flagellate,  non-sporogenous,  non- 
chromogenic,  non-aerogenic,  aerobic  and  optionally  anaerobic,  capsulated  ba- 
cillus, pathogenic  for  man  and  identical  with  Bacillus  pneumonia  of  Friedlander, 
except  that  it  stains  by  Gram's  method. 

A  peculiar  disease  of  the  nares,  characterized  by  the  formation 
of  circumscribed  nodular  tumors,  and  known  as  rhinosderoma,  is 
occasionally  seen  in  Austria-Hungary,  Italy,  and  some  parts  of 


Fig.  292. — Rhinoscleroma  (Courtesy  of  Mr.  Owen  Richards,  Cairo,  Egypt). 

Germany.  A  few  cases  have  been  observed  in  Egypt  and  a  few 
among  the  foreign-born  residents  of  the  United  States.  The  nodular 
masses  are  flattened,  may  be  discrete,  isolated,  or  coalescent,  grow 
with  great  slowness,  and  recur  if  excised.  The  disease  commences 
in  the  mucous  membrane  and  the  adjoining  skin  of  the  nose,  and 
spreads  to  the  skin  in  the  immediate  neighborhood  by  a  slow  invasion, 
involving  the  upper  lip,  jaw,  hard  palate,  and  sometimes  even  the 
*  "Wiener  med.  Wochenschrift,"  1882,  32. 


7i6 


Rhinoscleroma 


pharynx.  The  growths  are  without  evidences  of  acute  inflammation, 
do  not  usually  ulcerate,  and  upon  microscopic  examination  consist 
of  an  infiltration  of  the  papillary  layer  and  corium  of  the  skin,  with 
round  cells  which  in  part  change  to  fibrillar  tissue.  The  tumors 
possess  a  well-developed  lymph-vascular  system.  Sometimes  the 
cells  undergo  hyaline  degeneration. 

In  the  nodes,  von  Frisch  discovered  bacilli  closely  resembling  the 
pneumobacillus  of  Friedlander,  both  in  morphology  and  vegetation, 
and,  like  it,  surrounded  by  a  capsule.  The  only  differences  between 


Fig.  293. — Rhinoscleroma  (Courtesy  of  Mr.  Owen  Richards,  Cairo,  Egypt). 

the  bacillus  of  rhinoscleroma  and  Bacillus  pneumoniae  of  Friedlander 
are  that  the  former  stains  well  by  Gram's  method,  while  the  latter 
does  not;  that  the  former  is  rather  more  distinctly  rod- shaped  than 
the  latter,  and  more  often  shows  its  capsule  in  culturermedia. 

The  bacillus  can  be  cultivated,  and  cultures  in  all  media  resemble 
those  of  the  bacillus  of  Friedlander  (q.v.)  so  closely  as  to  be  almost 
indistinguishable  from  it.  The  chief  difference  lies  in  its  inability 
to  endure  acid  media  and  to  ferment  carbohydrates.  Even  when 
inoculated  into  animals  the  bacillus  behaves  much  like  Friedlander 's 
bacillus. 


Pathogenesis  717 

Inoculation  has,  so  far,  failed  to  reproduce  the  disease  either  in 
man  or  in  the  lower  animals. 

Pathogenesis. — The  bacillus  is  said  to  be  pathogenic  for  man  only, 
producing  granulomatous  formations  of  the  skin  and  mucous 
membranes  of  the  anterior  and  posterior  nares.  These  vary  in 


•   *  *  *~  *  X  fc         •  « 

*\V/»"-1   ^4^- 


Fig.  294. — Bacillus  rhinoscleromatis.     Pure  culture  on  glycerin  agar-agar.     Mag- 
nified 1000  diameters  (Migula). 

structure  according  to  age.  The  young  nodes  consist  of  a  loose 
fibrillar  tissue  composed  of  lymphocytes,  fibroblasts,  and  fibers. 
Some  of  the  cells  are  large  and  have  a  clear  cytoplasm  and  are 
known  as  the  cells  of  Mikulicz.  In  and  between  them  the  bacilli 
are  found  in  considerable  numbers.  The  older  lesions  consist  of  a 
firm  sclerotic  cicatricial  tissue. 


CHAPTER  XXXIII 
SYPHILIS 

TREPONEMA  (SPIROCHAETA)  PALLIDUM  (SCHAUDINN  AND  HOFFMANN) 

General  Characteristics. — A  non-chromogenic,  non-aerogenic,  anaerobic, 
minute,  slender,  closely  coiled,  flexible,  motile,  flagellated,  non-sporogenous, 
non-liquefying,  spiral  organism,  cultivable  upon  specially  prepared  media,  patho- 
genic for  man  and  certain  of  the  lower  animals,  staining  by  certain  methods  only 
and  not  by  Gram's  method. 

ALTHOUGH  syphilis  has  been  well  known  for  centuries,  its  specific 
cause  has  but  recently  been  discovered.  The  supposition  that  the 
disease  could  not  be  successfully  communicated  to  any  of  the 
lower  animals  was  supposed  to  explain  the  del  ay,  but  has  not  proved 
to  be  the  case,  for  in  spite  of  the  discovery  of  Metschnikoff  and 
Roux*  that  chimpanzees  could  be  successfully  inoculated  with  virus 
from  a  human  lesion,  the  confirmation  of  their  work  by  Lassarf  and 
others,  and  the  additional  discovery  of  Metschnikoff  and  Roux,J 
that  it  is  also  possible  to  infect  macaques  with  syphilis,  the  specific 
organism  was,  after  all,  discovered  for  the  first  time  in  matter 
secured  from  human  lesions. 

It  has  long  been  known  that  preputial  smegma  and  various 
ulcerative  lesions  of  the  generative  organs  contain  certain  spiral  or- 
ganisms. Bordet  studied  them  with  care,  expecting  to  prove  that 
they  were  concerned  with  the  etiology  of  syphilis,  but  it  remained 
for  Schaudinn  and  Hoffmann  §  to  discover  the  specific  micro- 
organism. They  point  out  that  there  are  two  separate  species  of 
spiral  organisms  commonly  found  in  ulcerative  lesions  of  the 
genitalia.  One  called  by  them  Spirochaeta  refringens  is  of  common 
occurrence,  the  other,  called  Spirochaeta  pallida,  later,  and  more 
correctly,  Treponema  pallidum,  is  found  only  in  syphilitic  lesions — 
and  is,  therefore,  their  probable  cause.  The  discovery  of  Tre- 
ponema pallidum  by  Schaudinn  and  Hoffmann  was  quickly  con- 
firmed by  Metschnikoff.||  It  is  now  universally  accepted  as  the 
cause  of  syphilis. 

Morphology. — The  organism  is  a  slender,  flexible,  closely  coiled 
spiral,  usually  showing  from  eight  to  ten  uniform  undulations,  but 
occasionally  being  so  short  as  to  show  only  two  or  three,  or  so  long 
as  to  show  as  many  as  twenty. 

*  "Ann.  de  PInst.  Pasteur,"  Dec.,  1903,  p.  8og. 
"Berliner  klin.  Wochenschrift,"  1903,  p.  1189. 
j  "Annales  de  PInst.  Pasteur,"  Jan.,  1904. 
§  "Deutsche  med.  Wochenschrift,"  May  4,  1905. 
l|  "Bull.  Acad.  de  med.  de  Paris,"  May  16,  1905. 
718 


Staining  719 

It  is  very  slender,  measuring  from  0.33  to  0.5  //,  in  breadth  to  3.5 
to  15.5  IJL  in  length  (Levaditi  and  Mclntosh). 

It  forms  no  spores.  Multiplication  seems  to  take  place  by 
longitudinal  division. 

It  is  motile,  and  when  observed  alive  with  a  dark  field  illuminator, 
can  be  seen  to  rotate  slowly  about  its  longitudinal  axis  at  the  same 
time  that  it  slowly  sways  from  side  to  side  with,  a  serpentine  move- 
ment. The  organisms  are  provided  with  flagella  at  one  end,  some- 
times one  at  each  end. 

Noguchi*  observed  two  types  of  treponema,  one  slender,  one 
stouter.  When  carried  through  culture  and  used  to  inoculate  rabbits 
their  differences  were  found  to  be  fairly  constant.  The  lesions  pro- 
duced in  rabbit's  testicles  varied  with  the  variety  of  organism  in- 
oculated, one  causing  a  diffuse,  the  other  a  nodular,  orchids.  He 
conjectured  that  the  distinction  may  be  of  value  in  explaining  certain 
obscure  points  in  human  syphilis. 

Staining. — /.  Films. — The  original  discovery  of  the  organism 
was  achieved  through  the  employment  of  Giemsa's  stain — a  modifi- 
cation of  the  Romanowsky  method.  But  by  this  method  the  organ- 
isms appeared  very  pale  and  not  very  numerous.  Goldhornf 
improved  it  as  follows: 

In  200  cc.  of  water,  2  grams  of  lithium  carbonate  are  dissolved  and  2  grams  of 
Merck's  medicinal,  Grubler's  BX,  or  Koch's  rectified  methylene  blue  added. 
This  mixture  is  heated  moderately  in  a  rice  boiler  until  a  rich  polychrome  has 
formed.  To  determine  this  a  sample  is  examined  in  a  test-tube  every  few  minutes 
by  holding  it  against  an  artificial  light.  As  soon  as  a  distinctly  red  color  is 
obtained,  the  desired  degree  of  heating  has  been  reached.  After  cooling  it  is 
filtered  through  cotton  in  a  funnel.  To  one-half  of  this  polychrome  solution  5  per 
cent,  of  acetic  acid  is  gradually  added  until  a  strip  of  litmus-paper  shows  above 
the  line  of  demarcation  a  distinct  acid  reaction,  when  the  remaining  half  of  the 
solution  is  added,  so  as  to  carry  the  reaction  back  to  a  low  degree  of  alkalinity.  A 
weak  eosin  solution  is  now  prepared,  approximately  0.5  per  cent.  French  eosin, 
and  this  is  added  gradually  while  the  mixture  is  being  stirred  until  a  filtered  sam- 
ple shows  the  filtrate  to  be  of  a  pale  bluish  color  with  a  slight  fluorescence.  The 
mixture  is  allowed  to  stand  for  one  day  and  then  filtered.  The  precipitate  which 
has  separated  is  collected  on  a  double  piece  of  filter-paper  and  dried  at  room  tem- 
perature (heating  spoils  it).  When  completely  dried  it  can  easily  be  removed 
from  the  paper  and  may  then  be  dissolved  without  further  washing  in  commercial 
(not  pure)  wood  alcohol.  The  solution  should  be  allowed  to  stand  a  day,  then 
filtered.  The  strength  of  this  alcoholic  solution  is  approximately  i  per  cent.  To 
use  the  stain,  one  drops  upon  an  unfixed  spread  enough  dye  to  cover  it,  permits  it 
to  act  for  three  or  four  seconds,  and  then  pours  it  off  and  introduces  the  glass 
slowly,  spread  side  down,  into  clean  water,  where  it  is  held  for  another  four  or  five 
seconds,  after  which  it  is  shaken  to  and  fro  in  the  water  to  wash  it.  It  is  next 
dried  and  examined  at  once  or  after  mounting  in  balsam.  The  spirochsetes 
appear  violet  in  color. 

GhoreyebJ  recommends  the  following  rapid  method  of  staining 
the  organism  in  smears.  A  thin  spread  is  to  be  preferred.  No  heat 
fixation  is  necessary: 

*  "Journal  of  Experimental  Medicine,"  1912,  xv,  No.  2,  p.  201. 

t  Ibid.,  1906,  vm,  p.  451. 

j  "Jour.  Amer.  Med.  Assoc.,"  May  7,  1910,  LIV,    No.  19,  p.  1498. 


720 


Syphilis 


1 .  Cover  the  smear  with  a  i  per  cent,  aqueous  solution  of  osmic  acid,  and  per- 
mit it  to  act  for  thirty  seconds.     This  solution  acts  as  a  fixative  and  mordant. 

2.  Wash  thoroughly  in  running  water. 

3.  Cover  the  smear  with  a  i :  100  dilution  of  Liquor  plumbi  subacetatis  (freshly 
prepared).     Permit  it  to  act  for  ten  seconds.     The  lead  unites  with  the  albumin 
to  form  lead  albuminate  which  is  insoluble  in  water. 

4.  Wash  thoroughly  in  running  water. 

5.  Cover  the  smear  with  a  10  per  cent,  aqueous  solution  of  sodium  sulphid. 
This  is  to  act  ten  seconds,  during  which  the  salt  transforms  the  lead  albuminate 
into  lead  sulphid  and  causes  the  preparation  to  turn  brown.     The  osmic  acid 
when  reapplied  causes  it  to  become  black. 

6.  Wash  thoroughly  in  running  water. 

The  whole  process  is  to  be  repeated  in  exactly  the  same  manner 
three  times,  the  washings  all  being  very  thorough.  The  preparation 
is  then  dried  and  mounted  in  Canada  balsam.  The  micro-organisms 
and  cellular  detritus  are  stained  black. 


Fig.  295. — Treponema  pallidum  in  the  periosteum  near  an  epiphysis  (Bertarelli). 

When  serum  from  a  primary  sore  or  other  syphilitic  lesion  is  treated 
by  these  methods,  a  number  of  the  spirochaeta  appear  well  stained 
and  a  number  very  palely  stained,  so  that  one  is  in  doubt  whether 
there  may  be  many  others  unstained,  and  this  seems  to  be  the  case, 
for  when  similar  smears  are  treated  by  other  methods  many  more 
can  be  found. 

Stern*  has  applied  the  method  of  silver  incrustation  to  the  ex- 
amination of  films  by  the  following  simple  procedure: 

Spreads  are  made  in  the  usual  manner,  dried  in  the  air,  and  then  for  a  few  hours 
in  an  incubating  ovenat37°C.  They  are  next  placed  in  a  10  per  cent,  solution  of 
nitrate  of  silver  in  a  colorless  glass  receptacle  and  allowed  to  rest  in  the  diffused 
daylight  of  a  comfortably  lighted  room  for  a  few  hours,  until  they  become  brown- 
ish metallic  in  appearance,  when  they  are  thoroughly  washed  in  water.  The  spi- 
rochaeta appear  black,  the  background  brownish. x 

*  "Berliner  klin.  Wochenschrift,"  1907,  No.  14. 


Staining  721 

Burri*  has  recommended  a  simple  and  rapid  method  of  demon- 
strating the  treponema  and  other  similar  organisms  by  the  use  of 
India  ink. 

A  drop  of  juice  is  squeezed  from  a  chancre  or  mucous  patch  and  mixed  with  a 
drop  of  India  ink  and  then  spread  upon  a  glass  slide  as  in  making  a  spread  of  a 
drop  of  blood.  As  the  ink  dries  it  leaves  a  black  or  dark  brown  field  upon  which 
the  spiral  organisms  stand  out  as  shining,  colorless,  and  hence  conspicuous 
objects.  Williams  uses  Higgins'  water-proof  ink,  and  Hiss  recommends  "chin- 
chin,  Giinther- Wagner  liquid  pearl  ink,  for  the  purpose. 

The  method  is  fairly  satisfactory  for  diagnosis  and  can  be  applied 
in  a  few  moments. 


Fig.  296. — Treponema  pallidum  impregnated  with  silver.  Film  prepared  from 
the  skin  of  a  macerated,  congenitally  syphilitic  fetus.  X  750  diameters  (Flex- 
ner).  The  dense  aggregation  of  organisms  may  indicate  agglutination. 

II.  Section. — Staining  the  organism  in  the  tissues  is  a  more 
difficult  matter,  for  the  Giemsa  stain  scarcely  shows  it  at  all.  Bert- 
arelli  and  Volpinof  tried  a  modification  of  the  van  Ermengen  method 
for  flagella  with  some  success,  but  there  was  no  real  success  until 
LevaditiJ  devised  his  methods  of  silver  impregnation. 

This  consists  in  hardening  pieces  of  tissue  about  i  mm.  in  thickness  in  10  per 
cent,  formol  for  twenty-four  hours,  rinsing  in  water,  and  immersing  in  95  per  cent, 
alcohol  for  twenty-four  hours.  The  block  is  then  placed  in  diluted  water  until 
it  sinks  to  the  bottom  of  the  container,  and  then  transferred  to  a  1.5  to  3  per  cent, 
aqueous  solution  of  nitrate  of  silver  in  a  blue  or  amber  bottle  and  kept  in  a  dark 

*  "Wiener  klin.  Wochenschrift,"  July  i,  1909. 
t  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Orig.,  1905,  XL,  p.  56. 
J  "  Compt.-rendu  de  la  Soc.  de  Biol.  de  Paris,"  1905,  LIX,  p.  326. 
46 


722  Syphilis 

incubating  oven  at  37°C.  for  from  three  to  five  days.  Finally,  it  is  washed  in 
water  and  placed  in  a  solution  of  pyrogallic  acid,  2  to  4  grams;  formol,  5  cc.; 
distilled  water,  100  cc.,  and  kept  in  the  dark,  at  room  temperature,  from  twenty- 
four  to  seventy-two  hours,  then  washed  in  distilled  water,  embedded  in  paraffin, 
and  cut.  The  treponemata  are  intensely  black,  the  tissue  yellow  brown.  The 
sections  are  finally  stained  with — (a)  Giemsa's  stain  for  a  few  minutes,  then 
washed  in  water,  differentiated  with  absolute  alcohol  containing  a  few  drops  of 
oil  of  cloves,  cleared  with  oil  of  bergamot  or  xylol,  or  (&)  concentrated  solution  of 
toluidin  blue,  differentiated  in  alcohol  containing  a  few  drops  of  Unna's  glycerin- 
ether  mixture,  cleared  in  oil  of  bergamot,  then  in  xylol,  and  mounted  in  Canada 
balsam. 

This  method  was  later  improved  by  Levaditi  and  Manouelian* 
by  the  addition  of  10  per  cent,  of  pyridin  to  the  silver  bath  just 
before  the  block  of  tissue  is  put  in,  and  by  using  for  the  reducing 
bath  a  mixture  of  pyrogallic  acid,  acetone,  and  pyridin. 

The  details  are  as  follows:  Fragments  of  organs  or  tissues  i  to  2  mm.  in  thick- 
ness are  fixed  for  twenty-four  to  forty-eight  hours  in  a  solution  of  formalin  10: 100, 
then  washed  in  96  per  cent,  alcohol  for  twelve  to  sixteen  hours,  then  in  distilled 
water  until  the  blocks  fall  to  the  bottom  of  the  container.  They  are  then  impreg- 
nated by  immersion  in  a  bath  composed  of  a  i  percent,  solution  of  nitrate  of  silver, 
to  which,  at  the  moment  of  employment,  10  per  cent,  of  pyridin  is  added.  Keep 
the  blocks  immersed  in  this  solution  at  room  temperature  for  two  or  three  hours, 
and  at  5o°C.  for  four  or  six  hours,  then  wash  rapidly  in  a  10  per  cent,  solution  of 
pyridin,  and  reduce  in  a  bath  composed  of  4  per  cent,  pyrogallic  acid,  to  which,  at 
the  moment  of  using,  10  per  cent,  of  pure  acetone  and  15  per  cent,  (total  volume) 
of  pyridin  are  added.  The  reduction  bath  must  be  continued  for  several  hours, 
after  which  the  tissue  goes  through  70  per  cent,  alcohol,  xylol,  paraffin,  and  sec- 
tions are  cut.  The  sections,  fastened  to  the  slide,  are  stained  with  Unna's  blue 
or  toluidin  blue,  differentiated  with  glycerin-ether,  and  finally  mounted  in  Canada 
balsam. 

Distribution. — -The  Treponema  pallidum  is  not  known  in  nature 
apart  from  the  lesions  of  syphilis.  It  has  now  been  found  in  all 
the  lesions  of  this  disease  and  in  the  blood  of  syphilitics  in  larger 
or  smaller  numbers.  The  discovery  has  greatly  modified  our  ideas 
of  the  tertiary  stage,  for  the  demonstration  of  the  organisms  in  its 
lesions  shows  them  to  be  undoubtedly  contagious.  The  greatest 
number  of  the  organisms  are  found  in  the  tissues — -especially  the 
liver — of  still-born  infants  with  congenital  syphilis. 

Cultivation. — The  cultivation  of  the  treponema  was  first  at- 
tempted by  Levaditi  and  Mclntosh,f  who,  deriving  the  organism 
from  an  experimental  primary  lesion  in  a  monkey  (Macacus  rhesus), 
carried  it  through  several  generations  in  collodion  sacs  inclosed  in 
the  peritoneal  cavity  of  other  monkeys  (Macacus  cynomolgus) 
and  in  the  peritoneal  cavity  of  rabbits.  They  were  unable,  how- 
ever, to  secure  the  treponema  in  pure  culture,  having  it  continually 
mixed  with  other  organisms  from  the  primary  lesion.  In  the 
mixture,  however,  they  were  able  to  maintain  it  for  generations 
and  study  its  morphology  and  behavior.  During  cultivation  its 
virulence  was  lost. 

Schereschewskyf    endeavored  'to    cultivate    the    treponema    by 

'  "  Compt.-rendu  de  la  Soc.  de  Biol.  de  Paris,"  1906,  LVIII,  p.  134. 
'  "Ann.  de  ITnst.  Pasteur,"  1907,  p.  784. 
J  "Deutsche  med.  Wochenschrift,"  1909,  xxxv,  835,  1260,  1652. 


Cultivation  723 

placing  a  fragment  of  human  tissue,  containing  it,  deep  down  into 
gelatinized  horse-serum.  The  treponema  grew  together  with  the 
contaminating  organism  and  no  pure  culture  was  secured.  Muhlens* 
and  Hoffmann,!  using  the  same  method,  succeeded  in  securing  pure 
cultures  of  the  treponema,  but  found  them  avirulent. 

Noguchi,J  taking  advantage  of  the  observations  of  Bruckner 
and  Galasesco§  and  Sowade,||  that  an  enormous  multiplication 
of  treponema  occurred  when  material  containing  it  was  inoculated 
into  the  rabbit's  testis,  performed  a  lengthy  series  of  cultivation 
experiments  with  the  enriched  material  thus  obtained.  The 
culture-medium  used  in  these  experiments  was  a  "  serum  water," 
composed  of  i  part  of  the  serum  of  the  sheep,  horse,  or  rabbit 
and  3  parts  of  distilled  water;  16  cc.  of  this  mixture  was  placed 
in  test-tubes  20  cm.  long  and  1.5  cm.  in  diameter  and  sterilized  for 
fifteen  minutes  at  ioo°C.  each  day  for  three  days. 

To  each  of  a  series  of  such  tubes  a  carefully  removed  fragment  of  sterile  rabbit's 
testis  was  added,  after  which  the  tubes  were  incubated  at  37°C.  for  two  days  to 
determine  their  sterility.  To  each  tube  the  material  from  the  inoculated  rabbit's 
testis,  rich  in  the  treponema,  is  added,  after  which  the  surface  of  the  medium  in 
each  receives  a  thick  layer  of  sterile  paraffin  oil.  As  the  most  strict  anaerobiosis 
is  necessary,  the  tubes  are  placed  in  a  Novy  jar,  the  bottom  of  which  contains 
pyrogallic  acid.  Noguchi  first  passes  H  gas  through  the  jar,  permitting  it  to 
bubble  through  the  pyrogallic  acid  solution  for  ten  minutes.  He  then  uses  a 
vacuum  pump  to  exhaust  the  atmosphere  in  the  jar,  and  lastly  permits  the  alka- 
line solution  (KOH)  to  flow  down  one  of  the  tubes  and  mix  with  the  pyrogallic 
acid. 

In  these  cultures  the  pallidum  grows  together  with  such  bacteria 
as  may  have  been  simultaneously  introduced.  To  secure  the 
cultures  free  from  these  bacteria  Noguchi  permitted  the  treponema 
to  grow  through  a  Berkefeld  filter,  which  for  a  long  time  held  back 
the  other  organisms.  Later  it  was  found  that  both  bacteria  and 
treponema  grow  side  by  side  in  a  deep  stab  in  a  serum-agar- tissue 
medium,  but  that  the  bacteria  grow  only  in  the  stab  or  puncture, 
whereas  the  treponema ta  grow  out  into  the  medium  as  a  hazy 
cloud.  By  cautiously  breaking  the  tube  and  securing  material  for 
transplantation  from  the  scarcely  visible  cloud,  the  organisms  may 
be  transplanted  to  new  media  and  pure  cultures  obtained. 

In  a  later  paper,  Noguchi**  details  the  cultivation  of  the  tre- 
ponema from  fragments  of  human  chancres,  mucous  patches,  and 
other  cutaneous  lesions.  The  medium  employed  is  a  mixture  of 
2  per  cent,  slightly  alkaline  agar  and  i  part  of  ascitic  or  hydrocele 
fluid,  at  the  bottom  of  which  a  fragment  of  rabbit  kidney  or  testis 
is  placed.  The  medium  is  prepared  in  the  tubes,  after  the  addi- 
tion of  the  tissue,  by  mixing  2  parts  of  the  melted  agar  at  5o°C.  with 


*  Ibid.,  1909,  xxxv,  1261. 

f  "Zeitschrift  fur  Hygiene  und  Infektionsk.,"  1911,  LXVIII,  27. 
J  "Journal  of  Experimental  Medicine,"  1911,  xrv,  99. 
§  "  Compt.-rendu  de  la  Soc.  de  Biol.  de  Paris,"  1910,  LXVIII,  648. 
||  "Deutsche  med.  Wochenschrift,"  191'!,  xxxvn,  682. 
'*  "Journal  of  Experimental  Medicine,"  1912,  xv,  i,  p.  90. 


724  Syphilis 

i  part  of  the  ascitic  or  hydrocele  fluid.  After  solidification  a  layer 
of  paraffin  oil  3  cm.  deep  is  added. 

A  considerable  number  of  tubes  should  be  prepared  at  the 
same  time  and  incubated  for  a  few  days  prior  to  use  to  determine 
sterility.  The  bits  of  human  tissue  are  snipped  up  with  sterile 
scissors  in  salt  solution  containing  i  per  cent,  of  sodium  citrate 
and  should  be  kept  immersed  in  this  fluid  from  the  time  of  securing 
to  the  time  of  planting,  so  as  not  to  become  dried.  A  bit  of  the 
tissue  should  be  emulsified  in  a  mortar  with  citrate  solution  and 
examined  with  a  dark  field  illuminator  to  make  sure  that  the  organ- 
isms to  be  cultivated  are  present. 

If  they  are  found,  and  the  material  shown  to  be  adapted  to  culti- 
vation, each  of  the  remaining  bits  of  tissue  is  taken  up  by  a  thin 
blunt  glass  rod  and  pushed  to  the  bottom  of  a  culture-tube  and 
into  each  tube  several  drops  of  the  emulsion  examined  are  intro- 
duced by  means  of  a  capillary  pipet,  also  inserted  deeply  into  the 
medium.  The  tubes  are  next  incubated  at  37°C.  for  two  or  three 
weeks.  In  successful  tubes,  in  which  the  medium  has  not  been 
broken  up  by  gas-producing  bacteria,  there  is  a  dense  opaque 
growth  of  bacteria  along  the  line  of  puncture,  and  a  diffuse  opales- 
cence  of  the  agar-agar  caused  by  the  extension  into  it  of  the  grow- 
ing treponemata.  A  capillary  tube  cautiously  inserted  into  the  opal- 
escent medium  withdraws  a  particle  that  can  be  examined  with 
the  dark  field  illuminator.  When  such  observation  shows  the  cause 
of  the  opalescence  to  be,  in  fact,  the  treponema,  the  tube  can  be 
cautiously  broken  at  some  appropriate  part  and  the  transplanta- 
tion made  from  the  opalescent  part  of  the  medium  to  fresh  appro- 
priate culture-media.  By  these  means,  after  a  few  trials,  pure 
cultures  of  treponema  were  secured. 

The  colonies  were  said  never  to  be  sharp,  but  always  faintly 
visible.  There  is  no  color  and  no  odor. 

I  By  inoculating  the  organisms  recently  secured  from  human 
lesions  (by  the  method  given)  into  monkeys  (Macacus  rhesus  and 
Cereopithicus  callitrichus)  Noguchi  was  able  to  produce  typical 
syphilis  of  the  monkey,  thus  showing  that  the  virulence  of  the 
organisms  was  not  lost  in  the  cultivation. 

Zinsser,  Hopkins  and  Gilbert*  found  it  possible  to  grow  Tre- 
ponema pallidum  in  massive  cultures  in  fluid  media.  They  em- 
ployed a  flask  with  a  long  slender  neck  like  a  "  specific  gravity  flask." 
The  flask  was  filled  with  slightly  acid  (0.2  to  0.8  per  cent,  acidity) 
broth  containing  sheep-serum,  ascitic  fluid,  horse-serum  or  rabbit- 
serum,  with  an  addition  of  autoclaved  and  hence  thoroughly  sterilized 
tissue  (kidney,  liver,  brain,  lung  or  heart  muscle)  and  covered  with 
sterile  neutral  paraffine  oil.  The  culture  contains  the  greatest 
number  of  organisms  after  three  weeks.  To  collect  them  for  mak- 
ing luetin,  etc.,  the  fluid  in  the  flasks  was  poured  into  tubes  and 
'"Journal  of  Experimental  Medicine,"  1915,  XXT,  No.  3,  p.  213. 


Pathogenesis  and  Specificity  725 

centrifugated  for  a  short  time  to  throw  down  scraps  of  the  nutrient 
tissue,  the  fluid  then  decanted  and  recentrifugated  rapidly  and  for 
a  longer  time  to  throw  down  the  micro-organisms. 

Pathogenesis  and  Specificity. — There  can  be  no  doubt  about  the 
causal  relation  of  Treponema  pallidum  to  syphilis.  It  is  unknown 
in  every  other  relation;  it  has  appeared  in  every  required  relation, 
and  thus  has  completely  fulfilled  the  laws  of  specificity  laid  down  by 
Koch.  Treponema  pallidum  is  not  only  pathogenic  for  man,  but, 
as  has  already  been  shown,  can  also  be  successfully  implanted  into 
chimpanzees,  macaques,  rabbits,  guinea-pigs,  and  other  experi- 
ment animals.  As  syphilis  is,  however,  unknown  under  natural 
conditions,  except  in  man,  it  may  be  looked  upon  as  a  human 
disease. 

The  organism  enters  the  body  through  a  local  breach  of  con- 
tinuity of  the  superficial  tissues,  except  in  experimental  and  con- 
genital infections,  where  it  may  immediately  reach  the  blood. 

In  ordinary  acquired  syphilis  the  point  of  entrance  shows  the 
first  manifestations  of  the  disease  after  a  period  of  primary  incuba- 
tion about  three  weeks  long,  in  what  is  known  as  the  primary  lesion 
or  chancre.  This  appears  as  a  papule,  grows  larger,  undergoes  super- 
ficial indolent  ulceration,  and  eventually  heals  with  the  formation 
of  an  indurated  cicatrix.  It  is  in  this  lesion  that  the  treponema 
first  makes  its  appearance.  From  this  lesion,  where  it  multiplies 
slowly,  it  enters  the  lymphatics  and  soon  reaches  the  lymph-nodes, 
which  swell  one  by  one  as  its  invasion  progresses.  During  this 
stage  of  glandular  enlargement  the  organisms  can  be  found  in  small 
numbers  in  juice  secured  from  a  puncture  made  in  the  gland  with 
a  hollow  needle.  This  period  of  primary  symptoms  (chancre  and 
adenitis)  includes  part  of  what  is  known  as  the  period  of  secondary 
incubation,  which  intervenes  between  the  appearance  of  the  chancre 
and  that  of  the  secondary  symptoms.  It  usually  lasts  about  six 
weeks.  During  this  time  the  organisms  are  multiplying  in  the 
lymph-nodes  and  occasionally  entering  the  blood.  What  fate 
the  organisms  meet  when  they  reach  the  blood  in  small  numbers  is 
not  yet  known,  but  the  slow  invasion  suggests  that  those  first  enter- 
ing are  destroyed,  and  that  it  is  only  when  their  numbers  are  great 
and  their  virulence  increased  that  they  suddenly  become  able  to 
overcome  the  defenses  and  permit  the  development  of  the  secondary 
symptoms.  The  period  of  secondary  symptoms  corresponds  to 
the  invasion  of  the  blood  by  the  parasite.  It  may  continue  from 
one  to  three  years,  during  which  time  the  patient  suffers  from 
general  symptoms,  fever,  etc.,  probably  due  to  intoxication  and 
local  symptoms,  such  as  alopecia,  exanthemata,  etc.,  due  to  local 
colonization  of  the  organisms.  At  the  end  of  this  period  a  partial 
immunity,  such  as  is  seen  in  other  infectious  diseases  (malaria), 
develops,  the  organisms  disappear  from  the  blood,  the  general  local 
and  constitutional  disturbances  recover,  and  the  patient  may 


726  Syphilis 

be  well.  Should  he  continue  to  harbor  some  of  the  micro-parasites, 
however,  there  may  be  an  insidious  sclerosis  of  the  blood-vessels 
and  parenchymatous  organs  consequent  upon  the  growth  and  mul- 
tiplication of  the  parasites,  or  there  may  be  after  many  years  a 
period  of  tertiary  symptoms  characterized  by  the  sudden  appear- 
ance of  severe  lesions  in  which  the  parasites  are  very  few  in  number. 
The  specific  organisms  are  present  in  juice  expressed  from  "the 
primary  lesion,  in  juice  from  the  buboes  and  enlarged  lymph-nodes; 
in  the  blood,  in  the  roseola,  and  all  of  the  secondary  lesions,  and 
sparingly  in  the  tertiary  lesions. 

In  congenital  syphilis  they  reach  the  fetus  from  the  ovum,  the 
spermatozoon,  or  the  blood  of  the  mother.  Prenatal  death  from 
syphilis  is  accompanied  by  lesions  in  which  enormous  numbers 
of  the  organisms  can  be  found,  and  furnishes  the  best  tissues  for 
their  experimental  demonstration  and  study. 

Lesions. — The  lesions  of  syphilis  are  so  numerous  that  the  reader 
is  referred  to  works  on  pathology  and  dermatology  for  satisfactory 
descriptions.  Here  it  may  suffice  to  say  that  though  diverse  in 
appearance  and  location,  they  have  certain  features  in  common. 
The  first  of  these,  and  that  which  naturally,  places  syphilis  among 
the  infectious  granulomata,  is  the  lymphocytic  infiltration  of  the 
tissues,  with  which  all  of  the  lesions  begin.  The  second  is  a  peculiar 
form  of  necrosis — slimy  when  superficial,  gummy  when  deep — with 
which  they  terminate.  The  third  is  a  tendency  toward  excessive 
cicatrization. 

Diagnosis. — It  is  now  possible  to  make  a  certain  and  early  diag- 
nosis of  syphilis  by  the  recognition  of  the  specific  organisms,  and  as 
the  difficulty  of  treatment  is  in  proportion  to  the  stage  at  which 
the  disease  arrives  before  treatment,  it  should  never  be  neglected. 

I.  Staining. — The    expressed    lymph    from    a    carefully    cleaned 
freshly  abraded  primary  lesion  can  be  stained  by  Giemsa's  method, 
or,  as  is  much  better  and  more  certain,  by  Stern's  method,  with 
nitrate  of  silver,  or  by  the  use  of  India  ink. 

II.  Dark-field  Examination. — For  those  who  possess  the  "dark- 
field  illuminator"  or  some  similar  apparatus  with  the  proper  lamp, 
direct  examination  of  the  fluid  expressed  from  the  lesions  can  be 
made,  and  the  living,  moving  organisms  recognized.     This  should 
be  the  quickest  method  of  diagnosis,  though  it  takes  practice. 

III.  Serum    Diagnosis. — Wassermann    and    Bruck    have  devised 
a  laboratory  method  of  making  the  diagnosis  of  syphilis  by  test- 
ing the  complement  fixing  power  of   the  patient's   serum.     This 
method,  now  known  as  the  "Wassermann  reaction,"  (q.v.)  is  given 
elsewhere  in  complete  detail. 

The  success  of  the  von  Pirquet  cutaneous  tuberculin  reaction  in 
assisting  the  diagnosis  of  tuberculosis  led  to  experiments  on  the 
part  of  a  number  of  investigators — Meirowsky,  Wolff-Eisner, 
Tedeschi,  Nobe,  Ciuffo,  Nicholas,  Favce,  and  Gauthier  and  Jodas- 


Diagnosis  727 

shon — -to  obtain  analogous  reaction  in  syphilis  by  applying  extracts 
of  syphilitic  tissues  to  the  scarified  epiderm  of  syphilitics.  Some 
reactions  were  observed,  but  Neisser  and  Bruck  found  that  similar 
reactions  occurred  when  a  concentrated  extract  of  normal  liver 
was  applied,  and  to  such  reactions  which  could  not  be  looked  upon 
as  specific,  Neisser  applied  the  term  "Umstimmung." 

After  having  successfully  achieved  the  cultivation  of  Treponema 
pallidum,  Noguchi*  resolved  to  try  the  effect  of  an  application  of 
an  extract  of  the  organisms  applied  to  the  skin,  in  the  hope  that  it 
might  provoke  a  reaction  useful  for  diagnosis.  To  this  end  he  pre- 
pared two  cultures,  one  in  ascitic  fluid  containing  a  piece  of  sterile 
placenta,  the  other  in  ascitic  fluid  agar  also  containing  a  piece  of 
placenta.  After  permitting  them  to  grow  under  strictly  anaerobic 
conditions  at  37°C.  until  luxuriant  development  occurred,  the  lower 
part  of  the  solid  culture  was  carefully  cut  off,  the  tissue  fragment 
removed,  and  the  rich  culture  carefully  ground  in  a  sterile  mortar, 
the  thick  paste  being  diluted  from  time  to  time  by  adding  a  little 
of  the  fluid  culture.  The  grinding  was  continued  until  the  emulsion 
became  perfectly  clear,  when  it  was  heated  to  6o°C.  for  one  hour 
upon  a  water-bath  and  0.5  per  cent,  of  carbolic  acid  added.  When 
examined  with  the  dark-field  illuminator,  40  to  100  dead  trepone- 
mata  could  be  seen  in  every  field.  Cultures  made  from  the  sus- 
pension remained  sterile  and  inoculation  into  rabbits'  testicles  was 
without  result. 

This  extract  of  the  treponema  culture  he  calls  luetin.  When  it 
was  applied  to  the  ear  of  a  normal  rabbit,  by  means  of  an  endermic 
injection  with  a  fine  needle,  an  erythema  appeared,  but  faded  within 
forty-eight  hours,  the  skin  resuming  its  normal  appearance,  but 
when  it  was  applied  to  the  ear  of  a  syphilized  rabbit,  at  the  end  of 
the  forty-eight  hours  the  redness  developed  into  an  induration  the 
size  of  a  pea  and  persisted  from  four  to  six  days,  disappearing  in 
ten  days.  In  one  case  a  sterile  pustule  developed. 

Luetin  was  tested  by  Noguchi  and  his  colleagues  upon  400  cases: 
146  of  these  were  controls,  177  syphilitics,  and  77  parasyphilitics. 
In  the  controls  there  was  erythema  without  pain  or  itching,  which 
disappeared  without  induration  within  forty-eight  hours.  In  the 
syphilitics  at  the  end  of  forty-eight  hours  there  was  an  induration 
in  the  form  of  a  papule  5  to  10  mm.  in  diameter,  surrounded  by  a 
zone  of  redness  and  telangiectasis.  This  slowly  increased  for  three 
or  four  days  and  became  dark  bluish  red.  It  usually  disappeared 
in  about  a  week.  Sometimes  the  papule  underwent  vesiculation 
and  sometimes  pustulation.  It  always  healed  kindly  without  in- 
duration. In  certain  cases  described  as  torpid,  the  erythema  cleared 
away  and  a  negative  result  was  supposed  to  have  resulted,  when 
suddenly  the  spots  lighted  up  again  and  progressed  to  vesiculation 
or  pustulation.  In  3  cases  there  were  constitutional  symptoms — 
*  "Journal  of  Experimental  Medicine,"  1911,  xin,  p.  557. 


728  Syphilis 

malaise,  loss  of  appetite,  and  diarrhea.  Noguchi  found  that  the 
reaction  is  specific,  that  it  is  most  striking  and  most  constantly 
present  in  tertiary,  latent  tertiary,  and  congenital  syphilis.  It, 
therefore,  forms  a  valuable  adjunct  to  diagnosis,  seeing  that  it  is 
most  evident  in  precisely  those  cases  in  which  the  Wassermann 
reaction  is  most  apt  to  fail.  A  few  early  cases  energetically  treated 
with  mercury  and  salvarsan  give  marked  reactions.  A  few  old 
cases  fail  to  give  it. 

SPIROCH^TA  REFRINGENS   (SCHAUDINN  AND  HOFFMANN) 

This  spiral  organism,  though  given  the  name  by  which  it  is  now  known  by 
Schaudinn  and  Hoffmann,  was  probably  first  described  by  Donne  under  the  name 
Vibrio  lineola.  It  is  probably  a  frequent  organism  of  the  skin  and  mucous  mem- 
branes, and  occurs  in  greatest  numbers  in  lesions  of  the  genitalia  because  of  the 
smegma  upon  which  it  customarily  lives.  It  is  present  in  most  primary  lesions 
of  syphilis,  but  is  no  less  frequently  found  in  non-syphilitic  lesions,  such  as  bal- 
anitis,  venereal  warts,  and  genital  carcinoma.  It  is  also  found  in  the  mouth  and 
on  the  tonsils.  According  to  Hoffmann  and  Prowazek*  it  is  not  entirely  harmless, 
but  has  a  pathogenic  action,  and  some  of  the  complicating  lesions  of  syphilis  as 
well  as  some  of  the  destructive  diseases  of  the  genitals  may  be  intensified  by  it. 
They  found  it  pathogenic  for  apes. 

Morphologically,  it  is  much  broader  than  Treponema  pallidum,  its  spiral  waves 
are  much  coarser  and  less  regular.  It  is  easy  to  stain  by  all  methods  and  is  hence 
easily  found.  It  has  been  cultivated  by  Noguchi.  f 

*  "Centralbl.  f.  Bakt.,"  etc.,  1906,  XLI. 

t  "Journal  of  Experimental  Medicine,"  May  i,  1912,  xv. 


CHAPTER  XXXIV 

FRAMBESIA  TROPICA  (YAWS) 

TREPONEMA  PERTENUE  (PALLIDULUM)  (CASTELLANI) 

THIS  peculiar,  specific,  infectious,  contagious,  chronic  febrile 
disease  of  the  tropics  is  characterized  by  the  appearance  upon  the 
skin  of  one  or  more  primary  papular  lesions — the  yaws — bearing 
some  semblance  to  raspberries,  and  by  subsequent  malaise,  fever, 
and  other  constitutional  disturbances.  These  are  later  followed  by 
the  appearance  of  a  second  crop  of  small  papules  which  grow  to  the 
size  of  a  pea  or  a  small  nut  or  may  grow  to  be  as  large  as  apples, 
which  become  covered  with  firm  scabs  and  gradually  cicatrize.  The 
patient  either  recovers  or  suffers  from  relapses  and  the  appearance  of 
further  crops  of  the  lesions.  The  duration  of  the  disease  varies  from 
a  few  weeks  to  several  years.  In  most  cases  the  constitutional  dis- 
turbances occur  only  at  the  period  preceding  the  development  of 
the  eruptions  and  for  a  short  time  afterward.  Little  children 
frequently  die;  older  children  and  adults  may  die  of  exhaustion  in 
case  extensive  lesions  with  marked  ulcerations  develop. 

The  patients  usually  recover  and  pigmented  areas  remain  for 
some  time  where  the  lesions  have  occurred. 

The  disease  appears  to  have  been  known  since  1525,  when  Oviedo 
became  acquainted  with  it  in  St.  Domingo.  It  has  always  been  very 
puzzling  because  it  bears  so  many  resemblances  to  syphilis;  but  the 
peculiar  raspberry-like  character  of  the  primary  lesion,  its  dispo- 
sition to  occur  upon  the  face,  mouth,  nose,  eyes,  neck,  limbs,  fingers, 
and  toes,  as  well  as  upon  the  genitals,  seem  to  point  in  another  di- 
rection, and  all  authorities  now  admit  that  it  is  not  syphilis,  but  an 
independent  disease. 

It  occurs  only  in  tropical  countries,  and  is  most  frequent  in 
equatorial  Africa  on  the  west  coast,  from  Senegambia  to  Angola. 
It  also  occurs  in  West  Soudan,  Algeria,  the  Nile  Valley,  and  in  the 
islands  about  the  east  coast  of  Africa.  It  has  been  seen  rarely  in 
South  Africa.  In  Asia  it  occurs  in  Malabar,  Assam,  Ceylon,  Bur- 
mah,  Siam,  Malay  Peninsula,  the  Indian  Archipelago,  Moluccas,  and 
China.  It  is  also  endemic  in  many  of  the  islands  and  archipelagos 
of  the  southern  Pacific. 

The  disease  rarely  makes  its  appearance  in  the  United  States,  and 
it  is  of  interest  to  know  that  Wood*  has  been  able  to  collect  nine 
such  cases  from  the  literature. 

*  "American  Journal  of  Tropical  Medicine,"  1915,  n,  No.  7,  p.  431. 

729 


730 


Frambesia  Tropica 


Specific  Organism. — The  cause  of  the  disease  was  unknown  until 
the  discovery  of  Treponema  pallidum,  which  opened  a  way  for  its 
investigation.  Castellani*  was  quick  to  seize  the  opportunity,  and 
in  the  same  year  in  which  Schaudinn  and  Hoffmann  discovered 
the  cause  of  syphilis,  announced  a  similar  organism  as  the  cause 
of  yaws.  At  the  time  of  discovery  he  called  it  Spirochaeta  pertenuis 
and  Spirochaeta  pallidula,  but  it  is  now  recognized  as  a  treponema 
and  is  called  Treponema  pertenue. 

Morphology. — The  organism  so  closely  resembles  Treponema 
pallidum  that  it  is  rather  by  knowing  the  source  from  which  the 
organism  was  derived  than  by  any  morphologic  distinctions  that 
the  two  are  separated.  It  is  said  to  be  a  little  shorter  than  T. 


L  »t         & 

Fig.  297. — Yaws  (photograph  by  P.  B.  Cousland,  M.  B.,  Swatow,  China) . 

pallidum,  measures  7  to  20  /*  in  length,  is  closely  and  regularly  coiled, 
and  is  said  to  have  rounded  ends. 

Staining. — It  stains  like  its  close  relative,  palely  with  most 
of  the  dyes.  The  silver  nitrate,  the  India  ink  methods,  and  the 
other  methods  of  staining  Treponema  are  all  appropriate,  both  for 
demonstrating  it  in  smears  from  the  lesions  or  in  sections  of  tissue. 

Cultivation. — The  organism  seems  not  yet  to  have  been  cultivated. 

Pathogenesis. — Castellani  f  has  succeeded  in  infecting  monkeys 
with  the  scrapings  from  yaws  papules.  The  infection  usually  re- 
sulted in  a  local  lesion,  though  there  was  also  a  generalized  infection, 
for  he  found  treponemata  everywhere  in  the  lymph-nodes.  When 
the  inoculation  material  was  filtered  and  all  of  the  organisms  re- 
moved, the  infectivity  was  destroyed.  Blood  and  splenic  substance 
from  the  infected  monkey,  containing  no  organisms  other  than  the 

*  "Brit.  Med.  Jour.,"  1905,  n,  282,  1280,  1330. 
t  "Jour,  of  Hygiene,"  1907,  vn,  p.  558. 


Diagnosis  731 

treponemata,  was  infective  for  other  monkeys.  When  monkeys 
successfully  inoculated  with  yaws  are  afterward  infected  with  syph- 
ilitic virus  they  are  not  immune.  On  the  other  hand,  monkeys  that 
have  successfully  been  inoculated  with  syphilis  are  not  immune 
against  yaws.  Levaditi  and  Nattan-Larrier*  differ  from  Castellani 
in  this  particular,  and  found  that  monkeys  infected  with  syphilis 
are  refractory  to  yaws.  Castellani  was  able,  by  means  of  com- 
plement-fixation tests,  to  detect  different  specific  antibodies  for 
syphilis  and  yaws.  Halberstadterj  has  successfully  infected 
orang-outangs. 

There  is  no  doubt  but  that  in  their  clinical  manifestations  and 
in  their  etiology  frambesia  and  syphilis  are  closely  related. 

Diagnosis. — In  addition  to  the  clinical  manifestations  which  are 
usually  quite  sufficient  for  diagnosis,  the  discovery  of  the  Treponema 
pertenue  is  of  assistance.  It  can  usually  be  found  without  difficulty 
by  expressing  the  serum  from  a  lesion  and  staining  it  by  any  of  the 
methods  recommended  for  Treponema  pallidum,  the  India-ink 
method  being  the  most  simple. 

The  Wassermann  reaction  is  always  positive  in  yaws,  hence  is  of 
no  use  for  purposes  of  differential  diagnosis. 

*  "Ann.  de  1'Inst.  Pasteur,"  1908,  xxn,  260. 

t  "Arbeiten  a.  d.  Kaiserl.  Gesund.,"  1907,  xxvi,  48. 


CHAPTER  XXXV 
ACTINOMYCOSIS 

ACTINOMYCES    BOVIS    (BOLLINGER) 

General  Characteristics. — A  parasitic,  pathogenic,  aerobic  and  optionally  anae- 
robic, non-motile,  non-flagellate,  non-sporogenous  (?),  liquefying,  pathogenic, 
branched  micro-organism,  belonging  to  the  higher  bacteria,  staining  by  ordinary 
methods  and  by  Gram's  method. 

In  1845  Langenbeck  discovered  that  an  infectious  disease  of 
cattle  known  as  " wooden  tongue"  and  " lumpy  jaw,"  and  later  as 
actinomycosis,  could  be  communicated  to  man.  The  observation, 
however,  was  not  published  until  1878,  one  year  after  Bellinger* 
had  discovered  the  actinomyces,  the  specific  cause  of  the  disease. 

Israelf  wrote  the  first  important  paper  upon  actinomycosis  as 
a  disease  of  man,  though  the  best  paper  on  the  subject  is  probably 


FIG.  298. — Bovine  actinomycosis. 

that  by  Bostrom,t  who  made  a  careful  study  of  the  microscopic 
lesions  of  the  disease. 

Its  first  manifestations  are  usually  found  either  about  the  jaw 
or  in  the  tongue,  and  consist  of  considerable  sized  enlargements 
which  are  sometimes  dense  and  fibrous  (wooden  tongue),  some- 
times suppurative  in  character.  In  sections  of  tissue  containing 
these  nodular  formations,  small  yellowish  granules  surrounded  by 
some  pus  can  usually  be  found.  These  granules,  when  examined  be- 
neath the  microscope,  consist  of  peculiar  rosette-like  bodies — the 
"ray-fungi"  or  actinomyces. 

Distribution. — 'The  actinomyces  is  best  known  as  a  parasitic 
organism  associated  with  actinomycosis.  That  it  occurs  rather 

*  "Deutsche  Zeitschrift  fur  Thiermedizin,"  1877. 

"Virchow's  Archives,"  1874-78. 

j  "Berl.  klin.  Wochenschrift,"  1885.  "Beitrage  zur  Path.  Anat.  und  zur  Allg. 
Path.,"  1890,  EX. 

732 


Morphology  733 

widely  in  nature  seems  to  be  indicated  by  the  fact  that  cases  of 
infection  have  been  known  to  occur  from  the  spines  of  barley  and 
other  cereals.  Berestnew*  succeeded  in  isolating  the  organisms  from 
hay  and  straw. 

Morphology. — A  complete  ray-fungus  consists  of  several  distinct 
zones  composed  of  different  elements.  The  center  is  composed  of 
a  granular  mass  containing  numerous  bodies  resembling  micro- 
cocci  or  spores.  Extending  from  this  center  into  the  neighboring 
tissue  is  a  radiating,  branched,  tangled  mass  of  mycelial  threads. 


•^•Vi'^xL 


*"*'3£mm 
,*•  s 

r-.     '* 


Fig.  299. — Colony  or  granule  of  actinomyces  in  a  section  through  a  lesion 
showing  the  Gram-stained  filaments  and  hyaline  material  and  also  the  pus- 
cells  surrounding  the  colony  (Wright  and  Brown). 

In  an  outer  zone  these  threads  are  seen  to  terminate  in  conspicuous, 
club-shaped,  radiating  forms  which  give  the  colonies  their  rosette- 
like  appearance.  The  clubs  are  inconspicuous  in  the  human  lesions 
of  the  disease. 

The  pleomorphism  of  the  organism  and  the  branched  network 
it  forms  class  it  among  the  higher  bacteria  in  the  genus  Actinomyces. 
When  the  clumps  formed  in  artificial  cultivations  of  the  parasite 
are  properly  crushed,  spread  out,  and  stained,  the  long  mycelial 
threads,  0.3-0.5  n  in  thickness,  occasionally  show  flask-  or  bottle- 
like  expansions — the  clubs — at  the  ends.  These  probably  depend 
*  "Centralbl.  f.  Bact.,"  etc.,  Ref.,  1898,  No.  24. 


734  Actinomycosis 

upon  gelatinization  of  the  cell-membrane  of  the  degenerating  para- 
site. The  club  is  one  of  the  chief  characteristics  of  the  organism. 
In  sections  of  tissue  the  radiating  filaments  are  very  distinct,  and 
the  terminal  clubs  are  all  directed  outward,  closely  packed  together, 
and  making  the  whole  mass  form  a  rounded  little  body  often  spoken 
of  as  an  "actinomyces  grain."  When  tissues  are  stained  first  with 
carmin  and  then  by  Gram's  method,  the  fungous  threads  appear 
blue-black,  the  clubs  red.  The  cells  of  the  tissues  affected  and  a 
larger  or  smaller  collection  of  leukocytes  form  the  surrounding  re- 
sisting tissue-zone. 

The  fungus  is  of  sufficient  size  to  be  detected  in  pus,  etc.,  by  the 
naked  eye.  As  it  usually  has  a  bright  yellow  color  it  is  not  in- 
frequently spoken  of  as  a  "sulphur  grain." 


Fig.  300. — Actinomyces  granule  crushed  beneath  a  cover-glass,  showing  radial 
striations  in  the  hyaline  masses.  Preparation  not  stained;  low  magnifying 
power  (Wright  and  Brown). 

Cultivation. — The  actinomyces  fungus  may  be  grown  upon  arti- 
ficial culture  media,  as  has  been  shown  by  Israel,*  Wolff,  and 
others. 

"The  granules,  preferably  obtained  from  closed  lesions,  are  first 
thoroughly  washed  in  sterile  water  or  bouillon  and  then  crushed  and 
disintegrated  between  two  sterile  slides.  If  one  is  working  with  a 
bovine  case  it  is  well  to  examine  microscopically  the  disintegrated 
material,  after  mixing  it  with  a  drop  or  two  of  bouillon  under  a  cover- 
glass,  to  see  if  filamentous  masses  are  present.  If  they  are  not,  or 
if  they  are  very  few,  proceed  no  further,  but  begin  again  with  another 
granule,  because  the  granules  in  bovine  lesions  sometimes  contain 
no  living  filaments  at  all,  but  may  be  composed  entirely  of  de- 
generated structures  from  which  no  growth  of  micro-organisms  can  be 

*  "Virchow's  Archives,"  cxv. 


Cultivation  735 

generated.  If  filaments  and  filamentous  masses  are  found  to  be 
present  in  the  granule,  then  the  disintegrated  products  of  the  granule 
are  to  be  transferred  by  means  of  the  platinum  loop  to  melted  i  per 
cent,  dextrose  agar-agar  contained  in  test-tubes  filled  to  a  depth  of 
7  or  8  centimeters  which  have  been  cooled  to  about  4o°C. 
The  material  is  to  be  thoroughly  distributed  throughout  the  melted 
agar-agar  by  means  of  the  loop,  and  the  tube  then  placed  in  the  in- 
cubator. Several  tubes  should  be  prepared.  At  the  same  time  a 
number  of  granules,  after  washing  in  sterile  water  or  bouillon,  should 
be  placed  on  the  sides  of  sterile  test-tubes  plugged  with  cotton  and  kept 
at  room  temperature  in  the  dark.  The  sugar-agar  tubes  inoculated 
as  above  described  should  be  examined  from  day  to  day  for  the 
presence  of  the  characteristic  colonies  in  the  depths  of  the  agar-agar. 
If  very  many  colonies  of  contaminating  bacteria  have  developed  in 
the  tubes,  it  will  probably  be  very  difficult  or  impossible  to  isolate 
the  specific  micro-organism.  If  there  are  few  or  no  contaminating 
colonies,  then  the  colonies  of  the  specific  organism  should  be  ex- 
pected to  develop  in  the  course  of  two  or  three  days  to  a  week. 
If  a  good  number  of  living  filaments  of  the  micro-organism  have 
been  distributed  throughout  the  agar,  the  specific  colonies  that 
develop  will  be  very  numerous  in  the  depths  of  the  agar,  especially 
throughout  a  shallow  zone  situated  about  5  to  12  mm.  below 
the  surface  of  the  agar-agar.  When  the  presence  of  the  char- 
acteristic colonies  has  been  determined,  slices  or  pieces  of  the  agar 
containing  colonies  are  to  be  cut  out  of  the  tube  by  means  of  a  stiff 
platinum  wire  with  a  flattened  and  bent  extremity.  A  piece  of  the 
agar-agar  is  to  be  placed  upon  a  clean  slide  and  covered  with  a  clean 
cover-glass.  It  is  to  be  examined  under  a  low  power  of  the  micro- 
scope, and  an  isolated  colony  selected  for  transplantation.  By 
obvious  manipulations,  under  continuous  control  of  microscopic 
observation,  the  selected  colony,  together  with  a  small  amount  of 
the  surrounding  agar-agar  is  to  be  cut  out,  care  being  taken  to 
be  sure  that  no  other  colony  is  present  in  the  small  piece  of  agar- 
agar  containing  the  colony.  The  small  piece  of  agar-agar  thus  cut 
out  should  not  have  a  greatest  dimension  of  more  than  2  mm.  The 
piece  of  agar-agar  is  then  transferred  from  the  slide  by  means  of  a 
platinum  loop  to  a  tube  of  sterile  bouillon  where  it  is  thoroughly 
shaken  up  to  free  it  from  any  adherent  bacteria.  If  there  be  any 
reason  to  believe  that  the  small  piece  of  agar  has  been  very  much 
contaminated  with  bacteria,  it  should  be  washed  in  a  second  tube  of 
bouillon,  then  the  piece  of  agar-agar  is  to  be  transferred  by  means  of 
the  platinum  loop  to  a  tube  of  melted  sugar-agar  cooled  to  4o°C. 
It  should  be  immersed  deeply  in  the  agar  and  the  tube  placed  in  the 
incubator.  If  the  colony  thus  transferred  to  the  agar-agar  is  capable 
of  growth,  in  the  course  of  some  days  it  will  have  formed  a  good- 
sized  colony  from  which  transplants  in  various  culture-media  may 
be  made." 


736 


Actinomycosis 


Fig.  301. — Colony  of  actinomyces  with  well-developed  "clubs"  at  the  periph- 
ery in  a  nodule  in  the  peritoneal  cavity  of  a  guinea-pig  inoculated  with  a  cul- 
ture from  another  guinea-pig.  Paraffin  section.  Low  magnification  (Wright). 
(Photograph  by  Mr.  L.  S.  Brown.) 


Fig.  302. — A  colony  of  actinomyces  in  a  nodule  twenty-eight  days  old  in  the 
peritoneal  cavity  of  a  guinea-pig  inoculated  with  a  culture  from  another  guinea- 
pig  (Bovine  case) .  The  "  clubs  "  are  well  developed  and  show  some  indications  of 
stratification.  Paraffin  section.  X  750  approx.  (Wright).  (Photograph  by 
Mr.  L.  S.  Brown.) 


Cultivation 


737 


From  such  anaerobic  cultures  the  micro-organism  can,  after  a  few 
generations,  be  made  to  grow  upon  the  surface  of  solid  media,  where 
it  invariably  forms  rounded  nodular,  elevated  masses. 


ABC 

Fig-  3O3- — Actinomycosis;  glycerin-agar  cultures:  A,  Discrete  rounded 
colonies  after  about  ten  days'  incubation  at  37°C.;  B,  limpet-shaped  colonies 
three  and  a  half  months  old;  C,  lichen-like  appearance  frequently  seen;  the 
growth  is  three  and  a  half  months  old  (Curtis). 

Blood-serum. — Upon  blood-serum  the  nodular  growths  present 
a  yellowish  or  rust-red  color,  and  are  surrounded  with  a  whitish  down 


738  Actinomycosis 

of  line  threads.  The  colonies  adhere  closely  to  the  culture-media 
and  are  so  firm  that  they  crush  with  difficulty.  If  the  surface  be 
scraped,  spores  and  fine  threads  may  be  secured.  If  the  mass  be 
crushed,  branched  filaments  may  be  secured.  The  colonies  become 
confluent  in  the  course  of  time,  and  a  thick  wrinkled  membrane 
is  produced.  The  growth  liquefies  blood-serum. 

Gelatin. — In  gelatin  puncture  cultures  an  arborescent  growth 
occurs  and  the  gelatin  is  liquefied. 

Agar-agar. — Upon  agar-agar  and  glycerin  agar-agar  the  growth 
is  similar  to  that  upon  blood-serum.  The  agar-agar  turns  brown 
as  the  culture  ages. 

Bouillon. — In  bouillon  the  growth  occurs  in  the  form  of  large 
granules  if  allowed  to  stand  quietly;  of  numerous  small  granules  if 
frequently  shaken  up.  The  granules  are  similar  in  structure  to 
those  formed  upon  the  dense  media.  The  bouillon  does  not  become 
clouded. 

Potato. — Upon  potato  the  growth  resembles  that  upon  blood- 
serum,  but  is  slower  in  developing.  The  color  is  reddish-yellow 
and  the  white  down  early  makes  its  appearance. 

Eggs. — The  organism  can  also  be  grown  in  raw  eggs,  into  which  it 
is  carefully  introduced  through  a  small  opening  made  under  aseptic 
precautions.  In  the  eggs  long,  branched  mycelial  threads  are 
formed. 

The  characteristic  rosettes  so  constantly  found  in  the  tissues  are 
never  seen  in  artificial  cultures. 

Metabolism. — There  seems  to  be  some  difference  of  opinion  as 
to  the  oxygen  requirement  of  actinomyces.  Israel,  Bostrom  and 
others  state  that  it  grows  best  when  provided  with  a  free  oxygen 
supply.  Wright  found  it  to  grow  best  under  anaerobic  conditions. 

It  does  not  ferment  sugar,  and  does  not  evolve  gas.  It  liquefies 
gelatin  and  blood-serum  but  does  not  coagulate  milk.  Some  strains 
seem  to  produce  a  small  quantity  of  orange-red  pigment. 

A  small  amount  of  soluble  toxin  appears  in  culture-filtrates. 

Temperature. — In  well  established  strains  accustomed  to  sap- 
rophytic  life,  growth  progresses  slowly  but  continuously  at  2o°C. 
(room  temperature).  Freshly  isolated  cultures  just  being  started 
will  only  grow  at  37°C.  Growth  ceases  at  a  point  between  45°C. 
and  5o°C.  Wright  found  the  organism  killed  after  an  hour  at  6o°C. 

Virulence.— When  the  actinomyces  is  grown  upon  artificial 
media  the  virulence  is  retained  for  a  considerable  time.  Different 
strains  show  varying  degrees  of  pathogenesis,  some  being  almost 
or  quite  non-pathogenic,  others  virulent.  The  difficulty  of  making 
successful  injections  of  the  laboratory  animals  limits  our  power  to 
accurately  gauge  the  virulence. 

Pathogenesis. — Actinomycosis  is  almost  peculiar  to  bovine 
animals,  but  sometimes  occurs  in  hogs,  horses,  and  other  animals, 
and  rarely  in  human  beings.  The  disease  can  with  difficulty  be 


Path'ogenesis 


739 


inoculated  into  experiment  animals,  the  introduced  fungi  cither  be- 
coming absorbed  or  encapsulated  by  connective  tissue  and  not  grow- 
ing. I  n  the  abdominal  cavities  of  rabbits  the  peritoneum,  mesentery 
and  omcntum  show  typical  nodules  containing  the  actinomyces 
rays  in  cases  of  successful  inoculation. 

Mode  of  Infection. — The  manner  by  which  the  organism  enters 
the  body  is  not  positively  known.     In  some  cases  it  may  be  by  direct 


Fig.  304. 


lomycosis  in  man  (Crookshank). 


inoculation  with  infectious  pus,  but  there  is  some  reason  to  believe 
that  the  organism  occurs  in  nature  as  a  saprophyte,  or  as  an  epiphyte 
upon  the  hulls  of  certain  grains,  especially  barley.  Woodhead  has 
recorded  a  case  where  a  primary  mediastinal  actinomycosis  in  the 
human  subject  was  apparently  traced  to  perforation  of  the  posterior 
pharyngeal  wall  by  a  barley  spikelet  accidentally  swallowed  by  the 
patient. 


740  Actinomycosis 

Cases  of  actinornycosis  are  fortunately  somewhat  rare  in  human 
medicine,  and  do  not  always  occur  in  those  brought  in  contact  with 
the  lower  animals.  The  fungi  may  enter  the  organism  through 
the  mouth  and  pharynx,  through  the  respiratory  tract,  through  the 
digestive  tract,  or  through  wounds. 

The  invasion  has  been  known  to  take  place  at  the  roots  of  carious 
teeth,  and  is  more  liable  to  occur  in  the  lower  than  in  the  upper 
jaw.  Israel  reported  a  case  in  which  the  primary  lesion  seemed  to 
occur  external  to  the  bone  of  the  lower  jaw,  as  a  tumor  about  the 
size  of  a  cherry,  with  an  external  opening.  Two  cases  of  the  dis- 
ease observed  by  Murphy,  of  Chicago,  began  with  toothache  and 
swelling  of  the  jaw.  A  few  cases  of  dermal  infection  are  recorded. 
Elsching*  has  seen  a  case  in  which  calcined  actinomyces  grains  were 
observed  in  the  tear  duct. 

When  inhaled,  the  organisms  enter  the  deeper  portions  of  the 
lung  and  cause  a  suppurative  broncho-pneumonia  with  adhesive 
inflammation  of  the  contiguous  pleura.  After  the  formation  of  the 
pleuritic  adhesions  the  disease  may  penetrate  the  newly  formed 
tissue,  extend  to  the  chest- wall,  and  ultimately  form  external  sinuses ; 
or,  it  may  penetrate  the  diaphragm  and  invade  the  abdominal  organs, 
causing  interesting  and  characteristic  lesions  in  the  liver  and  other 
large  viscera. 

Lesions. — 'The  degree  of  chemotactic  influence  exerted  by  the 
organism  seems  to  depend  upon  the  tissue  affected,  upon  the  pecu- 
liarity of  the  animal'  and  upon  the  virulence  of  the  organism.  When 
an  animal  is  but  slightly  susceptible,  and  especially  when  the  tongue 
is  affected,  the  disease  is  characterized  by  the  formation  of  cicatricial 
tissue — "wooden  tongue."  If,  on  the  other  hand,  the  animal  be 
highly  susceptible  and  the  jaw-bone  affected,  suppuration,  with  the 
formation  of  abscesses,  osteoporotic  cavities,  and  sinuses,  are  apt 
to  be  noticed.  This  form  of  the  disease  is  called  " lumpy  jaw"  in 
cattle. 

Before  the  nature  of  the  affection  was  understood  it  was  con- 
founded with  diseases  of  the  bones,  especially  osteosarcoma. 

From  the  tissues  primarily  affected  the  disease  spreads  to  the 
lymphatic  glands,  and  eventually  to  the  lungs.  Israel  has  pointed 
out  that  certain  cases  of  human  actinornycosis  begin  in  the  peribron- 
chial  tissues,  probably  from  inhalation  of  the  fungi. 

But  few  cases  recover,  the  disease  terminating  in  death  from  ex- 
haustion or  from  complicating  pneumonia  or  other  organic  lesions. 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  xvm,  p.  7. 


CHAPTER  XXXVI 
MYCETOMA,  OR  MADURA-FOOT 

ACTINOMYCES    MADURA    (VlNCENT) 

General  Characteristics. — A  non-motile,  non-flagellate,  sporogenous  (?), 
non-liquefying,  non-aerogenic,  chromogenic,  aerobic  and  optionally  anaerobic, 
branched,  parasitic  organism  belonging  to  the  higher  bacteria,  staining  by  ordi- 
nary methods  and  by  Gram's  method,  and  pathogenic  for  man. 

A  curious  disease  of  not  infrequent  occurrence  in  the  Indian  prov- 
ince of  Scinde  and  of  rare  occurrence  in  other  countries  is  known  as 
mycetoma,  Madura-foot,  or  pied  de  Madura.  Although  described 
as  peculiar  to  Scinde,  the  disease  is  not  limited  to  that  province,  but 
has  been  met  with  in  Madura,  Hissar,  Bicanir,  Delhi,  Bombay, 
Baratpur,  Morocco,  Algeria,  and  in  Italy.  In  America  less  than  a 
dozen  cases  of  the  disease  have  been  placed  on  record.  In  India  it 
almost  invariably  affects  natives  of  the  agricultural  class,  and  in 
nearly  all  cases  is  referred  by  the  patient  to  the  prick  of  a  thorn. 
It  usually  affects  the  foot,  more  rarely  the  hand,  and  in  one  instance 
was  seen  by  Boyce  to  affect  the  shoulder  and  hip.  It  is  more  com- 
mon in  men  than  in  women,  individuals  between  twenty  and  forty 
years  of  age  suffering  most  frequently,  though  persons  of  any  age 
may  suffer  from  the  disease.  It  is  insidious  in  onset,  no  symptoms 
being  observed  in  what  might  be  called  the  incubation  stage  of  a 
couple  of  weeks'  duration,  except  the  formation  of  a  nodular  growth 
which  gradually  attains  the  size  of  a  marble.  Its  deep  attachments 
are  indistinct  and  diffuse.  The  skin  over  it  becomes  purplish,  thick- 
ened, indurated,  and  adherent.  The  ball  of  the  great  toe  and  the 
pads  of  the  fingers  and  toes  are  the  points  most  frequently  invaded. 
The  lesions  progress  very  slowly,  and  in  the  course  of  a  few  months 
form  distinct  inflammatory  nodes.  After  a  year  or  two  the  nodes 
begin  to  soften,  break  down,  discharge  necrotic  and  purulent  mate- 
rial, occasioning  the  formation  of  ulcers  and  sinuses.  The  matter 
discharged  from  the  lesions  at  this  stage  of  the  disease  is  a  thin 
serum,  and  contains  occasional  fine  round  pink  or  black  bodies, 
similar  to  actinomyces  "grains,"  described,  when  pink,  as  resem- 
bling fish-roe ;  when  black,  as  resembling  gunpowder.  It  is  upon  the 
detection  of  these  particles  that  the  diagnosis  rests.  According  to 
the  color  of  the  bodies  found,  cases  are  divided  into  the  pale  or  ochroid 
and  melanoid  varieties. 

The  progress  of  the  disease  causes  an  enormous  enlargement  of  the 
affected  part.  The  malady  is  usually  painless. 


742 


Mycetoma,  or  Madura-foot 


The  micro-organismal  nature  of  the  disease  was  early  suspected. 
In  spite  of  the  confusion  caused  by  some  who  confounded  the  disease 
with  "  guinea- worm, "  Carter  held  that  it  was  due  to  some  indigenous 
fungus  as  early  as  1874.  Boyce  and  Surveyor  found  that  the  black 
particles  of  the  melanoid  variety  consisted  of  a  large  branching 
septate  fungus. 

Pale  Variety. — 'Kanthack  was  the  first  to  prove  the  identity  of 
the  fungus  with  the  well-known  actinomyces,  but  there  seems  to  be 
considerable  doubt  about  the  identity  of  the  species. 


Fig.  30S- — Mycetoma.    Dorsum  of  foot  showing  sinuses,  some  of  which  are  covered 
by  hard  brownish  crusts  (courtesy  of  Dr.  John  W.  Perkins). 

Morphology. — Under  the  microscope  the  organism  was  found  by 
Vincent*  to  be  branched  and  belong  to  the  higher  bacteria.  It 
consists  of  long,  branched  bacillary  threads  forming  a  tangled  mass. 
In  many-of  the  threads  spores  could  be  made  out.  He  was  unable 
to  communicate  the  disease  to  animals  by  inoculation. 

Cultivation. — -Vincent  succeeded  in  isolating  the  specific  micro- 
organism by  puncturing  one  of  the  nodes  with  a  sterile  pipette,  and 
*  "Ann.  de  1'Inst.  Pasteur,"  1894,  vni,  30. 


Lesions 


743 


cultivated  it  upon  artificial  media,  acid  vegetable  infusions  seeming 
best  adapted  to  its  growth.  It  develops  scantily  at  the  room  tem- 
perature, better  at  37°C. — in  from  four  to  five  days.  In  twenty  to 
thirty  days  a  colony  attains  the  size  of  a  little  pea. 

Bouillon. — In  bouillon  and  other  liquid  media  the  organisms  form 
little  clumps  resembling  those  of  actinomyces.  They  cling  to  the 
glass,  remain  near  the  surface  of  the  medium,  and  develop  a  rose- 
or  bright-red  color.  Those  which  sink  to  the  bottom  form  spheric 
balls  devoid  of  the  color. 

Gelatin. — -The  growth  in  gelatin 
is  not  very  abundant,  and  forms 
dense,  slightly  reddish,  rounded 
clumps.  Sometimes  there  is  no 
color.  There  is  no  liquefaction. 

Agar-agar. — Upon  the  surface  of 
agar-agar  beautiful  rounded,  glazed 
colonies  are  formed.  They  are  at 
first  colorless,  but  later  become  rose- 
colored  or  bright  red.  The  ma- 
jority of  the  clusters  remain  isolated, 
some  of  them  attaining  the  size  of 
a  small  pea.  They  are  usually  um- 
bilicated  like  a  variola  pustule,  and 
present  a  curious  appearance  when 
the  central  part  is  pale  and  the  pe- 
riphery red.  As  the  colony  ages  the 
red  color  is  lost  and  it  becomes  dull 
white  or  downy  from  the  formation 
of  aerial  hyphae.  The  colonies  are 
very  adherent  to  the  surface  of  the 
medium,  and  are  of  almost  carti- 
laginous consistence. 

Milk. — The  organism  grows  in 
milk  without  causing  coagulation. 

Potato.— Upon  potato  the  growth  of  the  organism  is  meager  and 
slow,  with  very  little  chromogenesis.  The  color-production  is  more 
marked  if  the  potato  be  acid  in  reaction.  Some  of  the  colonies 
upon  agar-agar  and  potato  have  a  powdery  surface,  either  from  the 
formation  of  spores  or  of  aerial  hyphse. 

Lesions. — Microscopic  study  of  the  diseased  tissues  in  myce- 
toma  is  not  without  interest.  The  healthy  tissue  is  sharply  separated 
from  the  diseased  areas,  which  appear  like  large  degenerated 
tubercles,  except  that  they  are  extremely  vascular.  The  mycelial 
or  filamentous  mass  occupies  the  center  of  an  area  of  degeneration, 
where  it  can  be  beautifully  demonstrated  by  the  use  of  appropriate 
stains,  Gram's  and  Weigert's  methods  being  excellent  for  the 
purpose.  The  tissue  surrounding  the  nodes  is  infiltrated  with  small 


Fig.  306. — Actinomyces  mad- 
urae  in  a  section  of  diseased  tis- 
sue (Vincent). 


744 


Mycetoma,  or  Madura-foot 


round  cells.  The  youngest  nodules  consist  of  granulation-tissue, 
whose  development  is  checked  by  early  coagulation-necrosis.  Giant- 
cells  are  few. 

Not  infrequently  small  hemorrhages  occur  from  the  ulcers  and 
sinuses  of  the  diseased  tissues;  the  hemorrhages  can  be  explained  by 
the  abundance  of  small  blood-vessels  in  the  diseased  tissue. 


Fig.  307. — Melanoid  form  of  mycetoma.  Section  showing  black  granules 
and  general  features  of  the  lesions  as  they  appear  under  a  low-magnifying  power. 
Zeiss  at  (James  H.  Wright). 


Fig.  308. — Melanoid  form  of  mycetoma,  showing  structure  and  appearance 
of  the  hyphae  of  the  mycelium  obtained  from  the  granules.  Zeiss  apochromat; 
4  mm.  (James  H.  Wright). 

The  Melanoid  Form  of  mycetoma  has  been  carefully  investigated 
by  Wright*  and  appears  to  depend  upon  an  entirely  different  micro- 
organism properly  classed  among  the  hyphomycetes.  It  is  probably 
identical  with  the  organism  described  by  Boyce  and  Surveyor. 

In  the  case  studied,  Wright  found  the  diseased  tissues,  consisting 

*  "Journal  of  Experimental  Medicine,"  1898,  vol.  m,  p.  421. 


The  Melanoid  Form 


745 


of  several  of  the  pads  of  the  toes,  to  be  either  translucent  and  myxo- 
matous  or  yellowish  and  necrotic  in  appearance.  The  black  granules 
were  embedded  in  the  tissue  and  appeared  mulberry-like  and  less 
than  i  mm.  in  diameter.  They  were  firm,  and  when  enucleated  and 
pressed  between  cover  and  slide  did  not  crush.  Only  after  digestion 
with  a  solution  of  caustic  potash  and  careful  teasing  could  the 


Fig.  309. — Melanoid  form  of  mycetoma.  Two  bouillon  cultures  showing  the 
powder-puff  ball  appearance.  In  one  the  black  granule  is  seen  in  the  center  of 
the  growth  (James  H.  Wright). 


Fig.  310. — Melanoid  form  of  mycetoma.  Potato  culture  of  the  hyphomycete 
obtained  from  the  granules.  The  black  globules  are  composed  of  a  dark  brown 
fluid  (James  H.  Wright). 

granules  be  resolved  into  the  hyphae  of  the  mold.  The  central  part  of 
the  granule  formed  a  reticulum,  with  radiating,  somewhat  clavate 
elements  projecting  from  it. 

In  sections  of  tissue  it  was  found  possible  to  stain  the  fungus  with 
Gram's  and  Weigert's  stains,  though  prolonged  washing  removed 
most  of  the  dye. 


746  Mycetoma,  or  Madura-foot 

Cultural  Characteristics. — Enucleated  granules  carefully  washed 
in  sterile  bouillon  and  then  planted  upon  agar-agar  afforded  cultures 
of  the  mold  in  25  out  of  65  attempts. 

The  growth  began  in  five  or  six  days,  appearing  on  solid  media 
as  a  tuft  of  delicate  whitish  filaments,  springing  from  the  black  grain, 
and  in  a  few  days  covering  the  entire  surface  of  the  medium  with  a 
whitish  or  pale  brown  felt- work.  Upon  potato  this  felt- work  sup- 
ports drops  of  brownish  fluid.  The  long  branched  hyphae  thus 
formed  were  from  3  to  8  M  in  diameter,  with  transverse  septa  in  the 
younger  ones.  The  older  hyphae  were  swollen  at  the  ends.  No  buds 
were  observed.  No  fruit  organs  were  detected.  In  fluid  media  the 
filaments  radiated  from  the  central  grain  with  the  formation  of  a 
kind  of  puff-ball.  Eventually  the  whole  medium  becomes  filled  with 
mycelia  and  a  definite  surface  growth  forms. 

The  general  characteristics  of  the  fungus  are  well  shown  in  the 
accompanying  illustrations  from  Wright's  paper. 


CHAPTER  XXXVII 
BLASTOMYCOSIS 

BLASTOMYCES  DERMATITIDIS  (GILCHRIST  AND  STOKES) 

THE  first  case  in  which  yeasts  or  blastomycetes  were  definitely 
connected  with  disease  seems  to  have  been  published  by  Busse.* 
He  observed  a  case  of  tibial  abscess  in  a  woman  thirty-one  years  of 
age,  who  died  about  a  year  after  coming  under  observation.  Post- 
mortem examination  showed  numbers  of  broken-down  nodular  for- 
mations upon  the  bones,  and  in  the  spleen,  kidneys,  and  lungs.  In 
all  of  these  lesions  he  found,  and  from  them  he  cultivated,  an  yeast, 
which,  when  introduced  in  pure  culture  into  animals — mice  and 
rats — proved  infective  for  them.  He  called  the  organism  Saccharo- 
myces  hominis,  and  the  affection  in  which  it  was  found  "Saccharo- 
mycosis  hominis." 

In  May,.  1904,  three  months  before  the  appearance  of  Busse's 
paper,  Gilchrist  exhibited  to  the  American  Dermatological  Associa- 
tion in  Washington,  microscopic  sections  from  a  case  of  cutaneous 
disease,  in  which  peculiar  bodies,  recognized  as  plant  forms,  were 
found.  After  the  appearance  of  Busse's  papers,  Gilchrist f  more 
fully  described  and  illustrated  his  findings,  calling  the  lesions 
"  blastomycetic  dermatitis."  Though  much  work  upon  pathogenic 
blastomycetes  has  been  published  and  pathogenic  forms  of  these 
micro-organisms  have  been  described  by  Sanfelice,{  Rabinowitsch,§ 
and  others,  the  chief  and  almost  the  sole  form  in  which  these  infec- 
tions make  their  appearance  is  a  dermal  infection  known  as 
' '  blastomycetic  dermatitis. ' ' 

The  infection  usually  begins  with  the  formation  of  a  papule  upon 
the  lace  or  one  of  the  extremities,  which  suppurates  and  evacuates 
minute  quantities  of  viscid  pus.  The  lesion  crusts  and  begins  to 
heal,  but  at  the  periphery  new  and  usually  minute  foci  of  suppuration 
occur,  so  that  while  the  original  lesion  tends  to  heal  very  slowly, 
with  much  cicatricial  formation,  it  is  always  spreading.  The 
progress  is  usually  slow,  and  Gilchrist's  first  case  spread  only  2 
inches  in  four  years. 

Though  the  progress  is  slow,  it  is  sure,  and  there  is  no  tendency 
to  spontaneous  recovery  in  most  cases,  nor  is  the  condition  modified 
by  treatment.  The  patients  may  die  from  intercurrent  disease  or 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1894,  xvi,  175. 
t  "Johns  Hopkins  Hospital  Reports,"  i,  269,  291. 

J  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1895,  xvn,  113,  625;xvm,  521; xx,  219 
§  "Zeitschrift  fiir  Hygiene,"  etc.,  1896,  xxi,  n. 

747 


748  Blastomycosis 

from  a  generalization  of  the  blastomycetic  infection,  which  not  in- 
frequently happens. 

After  the  work  of  Gilchrist  had  made  clear  the  symptomatology 
and  parasitology  of  the  disease,  a  number  of  other  cases  were  reported, 
and  Ricketts*  published  an  excellent  and  lengthy  summary  of  all 
the  cases  with  references  to  all  of  the  literature  up  to  that  date.  An- 
other very  interesting  paper  by  Montgomery,!  published  in  1902, 
contains  a  splendid  atlas  of  photographs  of  the  various  lesions  and  of 
the  cultures. 

In  addition  to  the  cutaneous  blastomycosis,  a  second  form  is 
also  occasionally  seen,  and  is  known  as  Coccidioidal  granuloma. 
It  seems  to  have  been  first  observed  by  Posadas  and  WernickeJ 
and  has  been  carefully  studied  by  Ophiils.§  In  this  form  of  the 
disease  the  lesions  are  in  the  internal  organs,  macroscopically  and 


Fig.  311. — Cutaneous  blastomycosis  (Montgomery). 

microscopically  resemble  tubercles,  and  can  only  be  differentiated 
from  them  by  the  presence  of  the  blastomyces  and  the  absence 
of  tubercle  bacilli.  The  lungs  may  be  affected,  and  Walker  and 
Montgomery  1 1  mistook  a  case  for  miliary  tuberculosis  of  the  lungs. 
According  to  Evans**  the  disease  seems  to  have  a  predilection  for 
the  central  nervous'  system. 

There  seems  to  be  little  reason  for  believing  that  there  is  any 
other  difference  than  that  of  distribution  between  the  blastomycetic 
dermatitis  and  the  blastomycetic  granuloma,  or  that  they  are  caused 
by  different  micro-organisms.  Regarding  the  organisms,  however, 
we  are  by  no  means  sure  that  there  are  not  several  species. 

*  "Jour.  Med.  Research,"  1901,  i,  373. 

•  "Jour.  Amer.  Med.  Assoc.,"  June  7,  1902,  i,  1486. 
J  "Jour,  de  Micro-organismen,"  1891,  xv,  14. 

§  "  Jour.  Experimental  Medicine,"  1905,  vi,  443.  Ophiils  and  Moffit,  "  Phila. 
Med.  Jour.,"  1900,  v,  1471. 

"Jour.  Amer.  Med.  Assoc.,"  1902,  xxxvm,  867. 
e  "Jour,  of  Infectious  Diseases,"  1909,  vi,  535. 


Cultivation  749 

Specific  Organism. — The  organism  presents  a  variety  of  appear- 
ances which  may  be  thus  brought  together:  First,  there  are  round 
and  elliptical  disk-like  bodies  that  some  regard  as  spores,  others  as 
the  primitive  or  yeast  form.  These  measure  10  to  30  p  in  greatest 
diameter,  are  distinctly  doubly  contoured,  highly  refracting,  and, 
though  sometimes  clear  and  transparent,  are  frequently  granular  and 
vacuolated.  From  them  buds  may  grow,  as  in  the  yeasts,  or  hypha 
may  form,  as  in  oidium.  In  artificial  cultivations  the  hypha  may 
form  a  tangled  mycelium. 

Staining. — The  organisms  are  usually  better  found  without 
staining.  They  do  not  stain  with  aqueous  anilin  dyes,  but  are  pene- 
trated by  warm  thionin,  alkaline  methylene-blue,  and  polychrome 
methylene-blue.  In  sections  of  tissue  stained  with  hematoxylon 


Fig.  312. — Giant  cell  from  a  cutaneous  lesion  in  blastomycosis,  showing  a  group 
of  blastomyces  (Montgomery). 

and  eosin  they  show  as  uncolored  circles;  with  thionin  and  alkaline 
methylene-blue  they  may  take  a  blue  color. 

Cultivation. — -The  organism  grows  readily  upon  artificial  media 
when  once  started,  but  the  primitive  culture  is  difficult  to  secure, 
because  the  cocci  and  other  associated  organisms  are  more  numerous 
than  the  blastomyces  and  outgrow  it.  It  seems  most  satisfactory 
to  first  infect  a  guinea-pig  with  the  organism  from  the  skin,  and  then 
start  the  cultivation  from  its  lesions  than  to  attempt  it  directly  from 
the  pus  from  human  dermal  lesions.  When  the  human  lesions  are 
internal,  pure  cultures  are  easily  started. 

Gilchrist  and  Stokes*  were  able  to  start  cultures  directly  from  the 
dermal  lesions.  Hiss  and  Zinsser  recommended  that  this  be  done 
*  "Journal  of  Experimental  Medicine,"  1898,  m,  53. 


750 


Blastomycosis 


by  greatly  diluting  the  culture  material,  so  as  to  separate  the 
contained  organisms  widely. 

Many  culture-media  prove  appropriate,  glycerin  agar-agar  and 
agar-agar  containing  i  per  cent,  of  dextrose  being  excellent.  When 
once  isolated  the  organism  is  easily  kept  growing  by  transplanting 
every  month  or  two. 

The  colonies  appear  in  a  few  days  as  small  round  hemispheric  dots 
with  numerous  prickles  about  the  surfaces.  Later  they  have  a 
moldy  appearance  from  the  development  of  aerial  hypha.  They  are 
almost  purely  aerobic,  those  on  the  surface  growing  well,  those 
deeply  seated  in  the  medium  scarcely  at  all. 

Agar-agar  Slants. — These  at  first  show  a  creamy  white  layer  that 
becomes  quite  thick,  and  is  moldy  and  fluffy  on  the  surface.  After 


Pt> 
- 4^)\          Tfe# 

Mr«ilr  i  ¥ 


Fig.   313. — Blastomyces   dermatitidis.     Budding  forms  and   mycelial  growths 
from  glucose  agar  (Irons  and  Graham,  in  "Journal  of  Infectious  Diseases"). 

a  few  weeks  the  agar-agar  begins  to  turn  yellow  and  later  may  be- 
come brown,  though  the  growth  itself  remains  white  and  unchanged. 
The  growth  is  firmly  attached  to  the  agar.  When  old,  the  growth 
wrinkles. 

Bouillon. — 'The  growth  is  not  luxuriant.  The  medium  is  not 
clouded  and  contains  fluffy  flocculi  of  stringy  viscid  material. 
Sugars  added  to  the  medium  may  be  fermented. 

Gelatin. — Growth  takes  place  with  aerial  hypha.  Liquefaction 
does  not  occur  or  is  very  slow. 

Potato. — Abundant  growth  with  aerial  hypha. 

Milk.— Not  coagulated,  not  acidified,  slowly  digested. 

There  is  some  difficulty  in  describing  the  cultures,  as  different 
authors  describe  them  quite  differently,  evidently  having  different 
organisms  or  different  strains  under  observation. 


Transmission 


Pathogenesis. — The  organisms  are  pathogenic  for  guinea-pigs, 
rabbits,  and  dogs,  in  which  an  abscess,  not  infrequently  followed  by 
a  generalized  infection,  takes  place. 

Lesions. — The  human  lesions  vary  somewhat.  Gilchrist's  first 
case  resembled  lupus  vulgaris,  other  cases  present  an  exaggeration 
of  the  ulcerative  element.  Cases  have  also  been  mistaken  for 


Fig.    314. — Cultures    of    Blastomyces    dermatitidis    upon    solid    culture- media 

(Montgomery). 

syphilis.  The  intractable  character  of  the  lesions  is  suggestive,  and 
the  finding  of  the  micro-organisms  in  the  viscid  pus  ispathognomonic. 

Upon  section  the  lesions  still  resemble  lupus  and  other  tuberculous 
lesions,  but  here  again  the  absence  of  tubercle  bacilli  and  the 
presence  of  the  blastomyces  enable  diagnosis  to  be  made. 

Transmission. — -The  disease  is  transmissible.  The  source  of  in- 
fection is  not  known. 


CHAPTER  XXXVIII 
RINGWORM 

TRICHOPHYTON  TONSURANS  (MALMSTEN) 

TINEA  trichophytina,  ringworm  of  the  scalp,  herpes  tonsurans, 
tinea  circinata,  ringworm  of  the  body,  herpes  circinatus,  tinea 
unguium,  onychomycosis,  tinea  imbricata,  herpes  desquamans,  tinea 
versicolor,  pityriasis  versicolor,  erythrasma,  etc.,  are  diseases  with 
well-marked  clinical  manifestations  and  differences,  all  of  which 
may  be  comprehended  under  the  general  term  dermatomycosis,  and 
are  caused  by  closely  related  forms  of  parasitic  fungi,  whose  generic 
and  specific  differences  are  matters  of  considerable  uncertainty. 

That  certain  of  the  diseases  affect  hairy  parts  and  others  hairless 
parts  of  the  body,  that  still  others  occur  about  the  nails,  and  that 
some  are  superficial  and  practically  saprophytic,  while  others  pene- 
trate more  deeply  and  are  undoubtedly  parasitic,  do  not  necessarily 
point  any  more  conclusively  to  essential  differences  in  the  infecting 
organisms  than  to  accidents  of  infection  and  variations  in  resisting 
power.  A  review  of  the  literature  leaves  the  student  with  a  deplor- 
able confusion  of  ideas,  and  a  feeling  that  the  synonomy  is  too  com- 
plicated and  the  use  of  terms  too  loose  to  permit  of  systematic  re- 
construction. 

The  discovery  of  micro-organisms  in  these  lesions  seems  to  have 
been  made  in  1842  by  Gruby,*  who  found  mycelial  threads  and 
spores  on  and  in  the  hairs,  and  in  1860  by  Hebra,f  between  the  epi- 
thelial cells.  The  organism  appears  to  have  been  called  Trichophy- 
ton  tonsurans  in  1845  by  Malmsten.  The  parasitology  of  all  of  the 
trichophyton  infections  was  thoroughly  studied  by  Sabouraud,J 
and  the  old  species,  Trichophyton  tonsurans,  divided  into  eleven  new 
species,  to  which  four  others  have  since  been  added,  so  that  there 
are  now  described,  with  or  without  justification,  Trichophyton 
crateriforme,  T.  acuminatum,  T.  violaceum,  T.  effractum,  T.  ful- 
matum,  T.  umbilicatum,  T.  regulare,  T.  pilosum,  T.  glabrum,  T. 
sulphureum,  T.  polygonum,  T.  exsiccatum,  T.  circonvulatum,  T. 
flavum,  and  T.  plicatili. 

In  general  it  is  customary  to  divide  the  organisms  into  two  groups, 
Trichophyton  microsporon  and  T.  megalosporon,  the  former  having 
large,  the  latter  small,  spores. 

*  "  Compt.-rendu,"  Paris,  1842,  xv. 

"Handbuch  der  spezulien  Path.  u.  Therapie  von  Virchow,"  in,  1860. 
j"Ann.  de  dermat.  et  de  syphilis,"  1892,  in;  1893,  rv;  1894,  v;  "Monats- 
hefte,"  1896,  576;  "La  Practique  dermatologique.     Trichophytie,"  1900. 

7S2 


Cultivation 


753 


Morphology. — 'The  trichophyton  parasites  form  delicate  mycelia 
composed  of  somewhat  slender  septate  hypha.  They  can  best  be 
observed  by  extracting  one  of  the  hairs,  including  its  root,  from  the 
diseased  area,  or  if  the  affection  be  upon  a  hairless  part  of  the  body, 
by  scraping  off  some  of  the  epiderm,  and  mounting  the  material 
between  a  slide  and  cover  in  a  drop  of  caustic  potash  solution  (20 
per  cent.).  Under  these  circumstances  the  spores  are  conspicuous 
and  so  numerous  as  to  give  the  impression  that  they  occur  in  rows 
in  a  kind  of  structureless  zooglea  upon  the  outside  of  the  hair.  In 
some  cases,  however,  especially  in  Trichophyton  megalosporon,  the 
hypha  may  be  observed  with  the  spores 
inside.  The  hypha  measure  from  2  to 
8  M  in  diameter,  are  usually  simple, 
and  rarely  divide.  The  spores  are 
from  2  to  3  M  in  diameter  in  the  Tri- 
chophyton microsporon  and  7  to  8  ju 
in  T.  megalosporon.  The  former  is  the 
more  common  upon  the  hairless,  the 
latter  upon  the  hairy,  portions  of  the 
skin. 

Cultivation. — The  organisms  may  be 
secured  in  pure  culture  without  much 
difficulty,  except  for  the  annoying  and 
almost  constant  presence  of  the  associ- 
ated bacteria  of  the  skin.  By  crushing 
the  hairs  and  scales  in  a  mortar  with 
some  dilute  KOH  solution,  and  then, 
after  thoroughly  distributing  the  spores 
through  the  alkaline  medium  which  dis- 
solves many  of  the  bacteria,  plates  can 
be  made  with  high  dilutions,  or  drops 
of  the  fluid  may  be  spread  over  potato, 
which  is  an  excellent  medium  for  the  culture. 

The  culture,  whether  upon  agar-agar,  glycerin  agar-agar,  glucose 
agar-agar,  gelatin,  or  potato,  occurs  in  the  form  of  a  tuft  of  white 
mycelial  filaments  with  aerial  hypha,  looking  like  a  tiny  white 
powder-puff.  Upon  the  surface  of  liquid  culture-media  the  growth 
appears  as  a  thick  wrinkled  pellicle  with  aerial  hypha  of  velvety 
appearance.  As  the  cultures  grow  older  the  lower  mycelial  growth 
becomes  yellowish  and  wrinkled,  but  the  aerial  hypha  maintain 
the  velvety  white  appearance.  Some  of  the  colonies  are 
mammillated,  some  are  crateriform.  Gelatin  is  liquefied,  the 
growth  floating  upon  the  surface  of  the  fluid.  As  the  cultures 
become  very  old  and  dry,  the  velvety  appearance  is  lost  and  the 
surface  becomes  powdery.  The  powder  detaches  only  when  the 
growth  is  touched,  and  does  not  shake  off. 


Fig.  315. — Invasion  of  a  hu- 
man hair  by  trichophyton:  A, 
Points  at  which  the  parasitic 
fungi  coming  from  the  epider- 
mis are  elevating  the  cuticle 
of  the  hair  and  entering  into 
its  substance.  Magnified  200 
diameters  (Sabouraud). 


754  Ringworm 

Pathogenesis. — The  trichophytons  are  pathogenic  for  man  and 
for  the  lower  animals.  They  spread  from  animal  to  animal  by  con- 
tact and  by  inoculation.  Men,  dogs,  cats,  horses,  sheep,  goats,  and 
swine  all  suffer  from  the  infection.  The  growth  of  the  hypha  be- 
tween the  epidermal  layers  causes  a  chronic  inflammation,  with 
hyperemia,  desquamation,  the  formation  of  some  papules,  and  oc- 


^^         ^r        .^^ 

Fig.   316.— Trichophyton   tonsurans.     Primary   cultures   twenty   days   old   on 
maltose  agar-agar.     Natural  size  (Sabouraud). 

casional  pustules.  The  invasion  of  the  hair-follicles  and  the  growth 
of  the  fungi  into  the  hairs  cause  them  to  become  fragile  and  break 
off,  as  well  as  to  loosen  and  drop  out. 

The  name  "barber's  itch"  results  from  the  frequent  transmission 
of  the  infection  by  the  barber's  razors.  The  disease  is  easily  trans- 
missible and  precautions  should  always  be  taken  to  prevent  its 
dissemination. 


CHAPTER  XXXIX 
FAVUS 

ACHORION    SCHONLEINII    (REMAK) 

FAVUS,  or  tinea  favosa,  is  a  chronic  and  destructive  form  of 
dermatomycosis  occurring  in  man  and  animals,  caused  by  a  fungus 
discovered  in  1839  by  Schonlein,*  and  called  in  his  honor  Achorion 
sdhonleinii  by  Remak  in  1845.  This  fungus  is  widely  distributed 
and  affects  mice,  cats,  dogs,  rabbits,  fowls,  and  men.  Among 
human  beings  it  usually  occurs  upon  the  scalp  and  other  hairy  parts 
of  the  body,  though  it  may  also  affect  the  hairless  portions  and  even 
attack  the  roots  of  the  nails.  It  is  more  frequent  in  children  than 
in  adults.  The  fungus  grows  vigorously  and  usually  forms  a  small 
sulphur  yellow  disk  about  the  base  of  a  hair.  The  edges  of  this 
detach,  become  everted,  and  the  whole  eventually  separates,  forming 
the  "scutulum,"  or  characteristic  lesion  of  the  disease.  The  reac- 
tion is  more  marked,  the  damage  done  greater,  and  the  disease  less 
tractable  than  in  other  forms  of  dermatomycosis. 

The  infection  seems  to  take  place  in  most  cases  by  way  of  the  hair- 
follicles,  and  the  mycelia  of  the  fungi  grow  into  and  about  the  hairs, 
invading  the  epiderm,  and  causing  atrophy  of  the  hair-follicles  by 
pressure.  Beneath  and  around  the  scutulum,  which  consists  chiefly 
of  the  fungi,  an  inflammatory  reaction  takes  place,  and  leukocytic 
invasion  and  ulceration  cause  the  scutulum  to  separate. 

Although  usually  confined  to  the  skin,  the  favus  infection  may  ex- 
tend to  the  mucous  membranes,  and  Kaposi  and  Kundratf  have 
reported  a  case  in  which  favus  fungi  were  found  to  have  invaded 
the  stomach  and  intestines. 

The  disease  runs  a  course  sometimes  extending  over  many  years. 
Crocker J  mentions  a  case  that  recovered  after  thirteen  years.  It 
may  remain  localized  upon  the  scalp  or  may  spread  itself  over  much 
of  the  skin  surface.  When  the  lesions  are  large  they  give  off  an 
odor  suggesting  that  peculiar  to  white  mice.  In  recovering,  the 
lesions  leave  considerable  cicatricial  scarring,  and  atrophy  of  hair- 
follicles,  sweat,  and  sebaceous  glands  is  inevitable. 

The  Specific  Organism. — The  Achorion  schonleinii  is  probably 
better  regarded  as  a  group  of  closely  related  organisms  than  as  a 
single  one.  Indeed,  Quincke  has  described  three  species,  though 
they  are  not  yet  generally  accepted. 

*  M  Oiler's  "Archiv,"  1839. 

f  "Ann.  de  Dermat.  et  de  Syph.,"  1895,  p.  104. 

j  "Diseases  of  the  Skin,"  Phila.,  1903,  p.  1276. 

755 


756 


Favus 


The  organism  can  be  studied  by  extracting  a  hair  and  examining 
it  in  KOH  or  NaOH  solution  (20  per  cent.),  or  by  teasing  a  scutulum 
in  the  same  medium  and  examining  with  a  low  power.  Sections  of 
the  skin  may  also  be  made  when  possible. 

The  fungus  resolves  itself  into  mycelial  threads,  and  spores.     The 


• « 


Fig.  317. — Favus.  Hairs  of  a  child  infected  with  Achorion  schonleinii.  A, 
Magnified  260  diameters;  B,  75  diameters.  The  large  rounded  bodies  are  drop- 
lets of  air  which  always  appear  after  treatment  with  40  per  cent,  potash  solution. 
The  linear  threads  are  the  fungi.  Some  are  without  spores,  others  contain  rows 
of  spores  (Sabouraud). 


scutulum  consists  of  masses  of  spores  at  the  center  and  about  the  hair, 
with  mycelia  containing  spores  at  the  edges.  From  the  mycelium 
hypha  are  given  off,  the  ends  being  knobbed  or  clavate. 

The  mycelial  threads  are  highly  refractile,  contain  granular  proto- 
plasm, and  are  of  varying    thickness.     Sometimes    the    terminal 


Cultivation 


757 


hypha  are  simple,  sometimes  they  fork,  the  ends  are  always  clavate. 
The  hypha  give  off  buds  at  right  angles  along  their  course. 

The  spores  are  oval,  doubly  contoured,  as  a  rule,  but  may  be 
round  or  pointed  and  more  or  less  polyhedral.  They  measure  3  to  8 
H  in  length  and  3  to  4  M  in  breadth.  They  form  the  great  central 
mass  of  the  scutulum,  which  is  the  oldest  part.  Together  with  them 
one  finds  a  number  of  detritus  granules,  fat-droplets,  and  occasional 
swollen  epidermal  cells. 

Cultivation. — The  cultivation  of  the  achorion  is  quite  easy  if 
care  be  used,  for  the  central  part  of  each  scutulum  contains  pure 
cultures  of  the  organism.  The  best  method  is  probably  that  of 
Krai,*  which  is  as  follows:  "A  good  deal  of  the  material  from  the 
scutula  is  rubbed  up  in  a  porcelain  mortar  dish  with  previously  heated 
diatomaceous  earth,  with  a  porcelain  pestle,  without  exerting  too 
much  pressure.  Melted  agar-agar  tubes  are  then  inoculated  with 


Fig.    318. — Achorion    schonleinii.  Fig.     319. — Achorion    schonleinii. 

Four  weeks  old  culture  upon  beer-  Pure   culture,    four    weeks    old,   on 

wort  agar-agar   (Kolle  and  Wasser-  beerwort      agar-agar      (Kolle     and 

mann).  Wassermann). 

two  or  three  loopfuls  of  the  crushed  material  and  poured  into  Petri 
dishes.  Greater  dilution  can  be  made  if  desired.  The  plates  are 
examined  after  forty-eight  hours. 

Cultures  may,  however,  be  directly  made  with  material  from  the 
center  of  a  scutulum.  Agar-agar  should  be  used,  as  the  cultures  grow 
best  at  the  body  temperature.  The  young  colonies  that  appear  in 
forty-eight  hours  can  easily  be  transplanted  by  fishing  under  a  lens. 

The  best  medium  was  found  by  Sabouraud  to  consist  of  maltose, 
4;  peptone,  2;  fucus  crispi,  1.5;  water,  100. 

As  the  colonies  eventually  become  quite  large  it  is  recommended 
that,  instead  of  tubes,  they  be  made  in  Erlenmeyer  flasks,  the  trans- 
planted little  colonies  being  placed  at  the  center  of  the  medium  con- 
gealed upon  the  bottom  of  the  flask. 

The  appearance  of  the  cultures  varies  considerably.     Plaut  gives 

*  See  Plaut,  in  Kolle  and  Wassermann's  "Pathogene  Mikroorganismen,"  I,  p. 
608. 


758  Favus 

two  principal  varieties:  (i)  The  waxy  type — &  yellowish  mass  of  a 
waxy  character  with  radiating  folds  and  a  central  elevation.  As  a 
rule  no  aerial  hyphae,  but  occasionally  short  aerial  hypha. 

(2)  The  downy  type — -this  forms  a  white  disk  with  a  velvety  or 
plush-like  covering  of  white  aerial  hypha.  Sometimes  instead  of 
white  the  color  is  yellowish  or  reddish.  A  marked  dimple  with  a 
smaller  elevation  usually  occurs  in  the  middle,  and  there  may  be 
radial  folds. 

Pathogenesis. — The  micro-organism  is  pathogenic  for  mice, 
rabbits,  cats,  dogs,  hens,  and  men,  in  all  of  whom  typical  scutula 
form.  Scutulum  formation  has  not  been  observed  in  guinea-pigs. 
The  disease  readily  spreads  from  animal  to  animal  by  direct  contact 
and  by  indirect  contact  by  the  use  of  combs,  hair-brushes,  and  simi- 
lar objects.  On  account  of  its  chronicity,  its  obstinacy,  its  disfig- 
urement, and  its  transmissibility  it  is  a  dangerous  disease,  and  one 
that  requires  prompt  isolation  of  the  patient  and  the  utmost  care  for 
the  prevention  of  contagion. 


CHAPTER  XL 
SPOROTRICHOSIS 

SPOROTRICHOSIS  is  a  somewhat  rare  disease  of  man,  caused  by 
various  members  of  a  genus  of  fungi  known  as  Sporotrichum  (Link- 
Saccardo).  The  first  occurrence  of  human  sporotrichosis  seems  to 
have  been  reported  by  B .  R.  Schenck.  *  The  isolated  micro-organism 
in  this  case  was  carefully  studied  and  later  was  found  to  be  identical 
with  a  micro-organism  isolated  from  another  case  of  somewhat  simi- 
lar character  studied  by  Hektoen  and  Perkins,  f  who  described  it  as 
Sporotrichum  schenckii.  In  1903  de  Beurmann  {  and  his  associates 
took  up  the  subject  in  France,  and  Lutz  and  Splendore§  in  Brazil, 
and  new  cases  were  reported.  On  Aug.  8,  1908,  the  writer  of  an 
editorial  in  the  Journal  of  the  American  Medical  Association  was 
able  to  give  references  to  14  cases  of  the  disease.  In  1912  Ruediger|| 
was  able  to  collect  57  cases  that  had  occurred  in  the  United  States. 
In  1912  de  Beurmann**  reported  that  more  than  200  cases  had  been 
put  on  record  since  the  beginning  of  his  work  in  1903.  It  will  thus 
be  seen  that  the  recognition  of  the  cause  of  the  disease  and  the  im- 
provement in  diagnosis  that  followed  it  have  made  possible  the 
detection  of  many  cases  of  a  disease  not  recognized  until  1900. 

According  to  de  Beurmann  who  has  shown  great  interest  in  the 
affection  and  prosecuted  its  study  with  much  industry,  the  known 
organisms  of  the  Sporotrichum  group  comprise  the  following: 

Sporotrichum  schencki. 

Sporotrichum  beurmanni. 

Sporotrichum  beurmanni  var.  asteroides  (Splendore). 

Sporotrichum  beurmanni  var.  indicum  (Castellani). 

Sporotrichum  jeanselmei. 

Sporotrichum  gougerati. 

Specific  Organism. — The  Sporotrichum  is  characterized  by  a 
filamentous  spore-bearing  mycelium.  The  filaments  are  fine,  meas- 
uring about  2  M  in  diameter,  partitioned,  colorless,  much  branched 
and  tangled.  The  chief  feature  is  the  occurrence  of  the  spores  which 
are  situated  along  the  length  of  the  recumbent  filaments  either  on 

*  "  Bulletin  of  the  Johns  Hopkins  Hospital,"  Dec.,  1898. 
t  "Journal  of  Experimental  Medicine,"  1900,  1901,  v,  77. 
j  Ann.  de  Dermatologie  et  Syphilographie,  1906,  538. 
§  "Centralbl.  f.  Bakt.,  etc.,"  1907,  XLV,  Orig.,  632. 
|]  "Jour,  of  Infectious  Diseases,"  1912,  xi,  193. 
**  "British  Medical  Journal,"  Aug.  10,  1912,  n,  2900. 

759 


Sporotrichosis 


their  extremities  or  on  branches.  They  are  arranged  in  cylindrical 
cuffs  about  10  M  in  size  and  in  glomeruli.  As  a  matter  of  fact  the 
spores  are  readily  isolated  from  one  another.  They  arise  one  by  one 
in  variable  numbers  along  the  mycelium,  but  as  a  rule  in  very  large 
quantity  in  each  segment  of  the  thallus.  There  is  no  apparent  order 
in  their  arrangement.  So  long  as  it  remains  on  the  filament  the  spore 
appears  pear-shaped.  It  is  attached  by  a  very  fine  sterigma,  from 
1-2  M  in  length  and  from  0.5  /-t  in  width.  When  shed,  the  spore  is  oval. 
Its  dimensions  vary  from  3-5-6  M  in  length  and  from  2-3-4  AI  in 
breadth.  The  form,  the  distribution  and  the  brown  color  of  the 
spores  and  their  fructification  in  the  form  of  cylindrical  cuffs,  arranged 
in  branches  at  the  extremities  of  the  filaments,  constitute  together 


Fig.  320. — Sporothrix  schenckii. 
Margin  of  living  hanging-drop  cul- 
ture (gelatin)  X  about  150  (Hektoen 
and  Perkins  in  "Tour,  of  Exper. 

Med.")- 


Fig.  321. — Sporothrix  schenckii. 
Slant  culture  on  glucose  agar,  eight 
days  old  (Hektoen  and  Perkins,  in 
"Jour,  of  Exper.  Med."). 


with  the  original  substratum  of  the  fungus,  a  group  of  characters 
which  differentiates  Sporotrichum  beurmanni  sharply  from  all  other 
sporotrichs  (Matruchat). 

Hektoen  and  Perkins  thus  describe  Sporotrichum  schenckii:  The 
threads  of  the  mycelium  are  seen  to  be  doubly  contoured;  the  proto- 
plasm is  somewhat  granular  and  interrupted  at  fairly  regular  in- 
tervals by  transverse  septa;  the  diameter  of  the  threads  varies  some- 
what, the  average  being  about  2  // ;  the  branches  are  not  frequent  and 
do  not  bear  any  fixed  relations  to  the  septa.  In  the  hanging-drop 
cultures  the  relations  of  the  conidia  to  the  mycelium  are  very  nicely 
shown.  The  spore-bearing  branches  which  grow  out  in  a  radiating 
manner  from  the  central  feltwork,  are  commonly  tipped  by  a  cluster 


Staining 


761 


of  from  three  to  six  or  more  conidia,  which,  in  the  case  of  the  larger 
cluster,  are  attached  by  the  smaller  end  to  the  slightly  expanded 
extremity  of  the  branch.  Similar  ovate  buds  also  arise  from  the 
sides  of  the  hyphae  at  shorter  or  longer  intervals.  The  spores  are  also 
doubly  contoured  and  granular,  resembling  very  much  yeast  cells. 
These  various  features  are  well  shown  in 
the  photographs  on  the  accompanying 
plate.  The  attachment,  by  means  of  the 
short  pedicles  of  the  spores  to  the  threads, 
is  very  easily  severed  as  shown  by  the 
difficulty  in  obtaining  stained  preparations 
with  the  spores  in  situ.  When  placed  in 
the  hanging  drop,  the  conidia  grow  out  into 
one  or  more  straight  germ  tubes  which 
spring  from  either  or  both  ends  or  from.' 
the  side.  These  embryonal  threads  again 
give  rise  to  lateral  or  terminal  buds,  which 
in  all  particulars  resembles  the  spores  and 
some  of  which  form  branching  spore-pro- 
ducing threads,  so  that  in  the  early  stages 
very  peculiar-looking  bodies  are  produced. 

In  the  tissues  and  in  the  pus  from  the 
lesions  of  the  disease  the  parasites  have 
quite  a  different  appearance,  assuming  a 
short  oblong  form  like  a  thick  short  ba- 
cillus 3-5  fj,  in  length  and  2-3  ^  broad, 
basophilic,  finely  granular  and  surrounded 
by  a  very  delicate,  colorless  membrane,  de 
Beurmann  has  watched  the  growth  of  this 
degraded  form  of  the  parasite  into  the 
filamentous  and  spore-bearing  form,  in  ar- 
tificial culture. 

Staining. — The  micro-organism  is  much 
better  examined  in  the  fresh  and  living  con- 
dition than  dried  and  stained  as  it  greatly 
changes  in  appearance  through  shrinking. 
It  does  stain,  however,  with  the  usual  dyes, 
and  retains  Gram's  stain  except  when 
the  alcohol  washing  is  unduly  prolonged. 

Cultivation,  Colonies. — Upon  agar-agar,  at  the  end  of  about 
forty-eight  hours,  the  colonies  appear  elevated,  whitish,  with  feathery 
fringes  and  some  filamentous  downgrowths  into  the  medium. 
Upon  gelatin  the  downward  growth  results  in  liquefaction  and  the 
growing  colonies  sink  below  the  surface. 

Agar-agar. — Along  the  needle  track  made  by  a  stroke  culture,  a 
grayish  granular  slightly  elevated  line  with  feathery  edges  forms  in 
forty-eight  hours  and  in  seventy-two  hours  assumes  the  form  of  a 


Fig.  322. — Abscesses 
caused  by  sporothrix 
schenckii.  Arm  of  patient 
showing  ulcers  and  scars, 
at  a  late  stage  of  the  lesions 
(Hektoen  and  Perkins,  in 
"Jour,  of  Exper.  Med."). 


762  Sporotrichosis 

band  with  numerous  transverse  wrinkles;  in  a  couple  of  days  more 
"the  surface  becomes  more  markedly  corrugated  and  looks  like  a 
chain  of  mountains  on  a  map."  About  the  seventh  day,  the 
growth,  which  has  increased  in  thickness,  becomes  light  brownish  in 
color,  the  margins  being  smooth  and  wavy  and  marked  by  shallow 
transverse  grooves.  Still  later  the  growth  becomes  dark  brown, 
wrinkled  and  covered  by  a  delicate  fuzz.  The  agar-agar  becomes 
brown. 

Gelatin. — -In  gelatin  punctures  the  growth  is  confined  to  the  upper 
strata.  Lateral  branches  are  sent  out  from  the  needle  track.  A  sur- 
face felt-like  mass  of  mycelial  threads  forms  beneath  which  the  gela- 
tin liquefies.  The  surface  growth  sinks  into  the  liquid  medium. 

Blood-serum. — -The  growth  is  somewhat  like  that  on  agar-agar 
but  not  so  massive.  It  is  apt  to  be  covered  by  a  white  down. 

Bouillon. — -The  growth  which  is  fairly  abundant,  is  in  flakes  and 
tufts,  shreds  and  filaments  that  settle  to  the  bottom  or  cling  to  the 
sides.  A  white  surface  film  is  apt  to  cover  the  liquid.  No  fermenta- 
tion occurs  in  sugar  bouillon. 

Potato. — 'Upon  potato  tufts  form  in  twenty-four  hours.  These 
have  a  brownish-gray  color  and  soon  become  raised,  wrinkled,  and 
frosted.  The  potato  is  darkened. 

Milk. — -The  growth  is  scanty  and  owing  to  the  opacity  of  the 
medium,  difficult  to  see.  Litmus  milk  is  not  acidified.  There  is 
no  coagulation. 

Vital  Resistance. — 'The  optimum  temperature  is  about  37°C.  The 
organism  grows  slowly  at  room  temperature  but  in  the  end  attains 
pretty  much  the  same  magnitude  as  those  kept  in  the  thermostat. 
The  death  point  is  55°C.  for  one  hour.  Hektoen  and  Perkins  found 
S.  schenckii  killed  in  four  and  one-half  minutes  at  6o°C. 

Metabolic  Products. — 'The  organism  produces  no  curdling  or 
proteolytic  ferments  for  milk  or  blood-serum.  It  does,  however, 
liquefy  gelatin.  It  grows  aerobically  or  anaerobically,  but  under  the 
latter  conditions  it  does  not  produce  acid  or  ferment  sugars,  or 
evolve  gas.  No  indol  is  formed.  It  has  a  remarkable  tolerance 
for  acid  media.  Page,  Frothingham  and  Paige*  found  that  it  grew 
well  in  media  at  least  six  times  as  acid  as  those  ordinarily  employed 
for  bacteria.  They  also  found  that  the  organism  does  produce  acid 
in  media  containing  dextrose. 

Distribution  in  Nature. — According  to  de  Beurmann,  the  sporotri- 
chum  is  a  widely  distributed  micro-organism  in  nature.  It  has  been 
found  on  green  vegetables,  upon  bark,  thorns,  potatoes,  various  im- 
plements, in  the  soil,  and  in  infected  insects. 

Pathogenesis. — 'The  sporotrichum  is  pathogenic  for  men,  horses, 
rats,  dogs,  and  white  mice. 

It  would  seem  as  though  the  rarity  of  its  occurrence  as  a  patho- 
genic agent  signified  that  it  was  by  no  means  easy  for  it  to  effect  the 
*  "Jour-  Med.  Research,"  1910,  xxm,  p.  129. 


Pathogenesis  763 

invasion  of  the  animal  body.  However,  de  Beurmann  mentions  a 
man  wounded  in  the  forehead  by  a  coster's  awl  whom  he  believed 
to  have  been  infected  by  a  cap,  used  to  conceal  the  untreated  wound, 
that  usually  lay  on  the  fruit  and  vegetables  that  filled  his  barrow; 
a  market  woman  infected  by  the  salad  that  she  was  in  the  habit  of 
handling  all  day.  Dominici  and  Duval  report  a  case  following  a  cut 
inflicted  while  peeling  a  potato;  Saint-Girons,  a  case  following  the 
prick  of  a  thorn  of  a  barberry  bush.  A  patient  of  Lutz's  was  inocu- 
lated through  the  bite  of  a  cat;  one  of  Wyse-Lauzun's  through  the 
bite  of  a  parrot.  Perkins'  case  was  that  of  a  child  that  had  abraded 
a  finger  with  a  hammer,  de  Beurmann  found  the  organism  in  the 
pharynx  of  healthy  persons  "carriers,"  whose  saliva  might,  therefore, 
be  infectious.  He  believes  that  infection  may  take  place  through  the 
hair-follicles;  that  the  healthy  skin  may  be  penetrated,  and  that  the 
healthy  gastro-intestinal  mucosa  may  be  penetrated. 

Lesions. — -The  seat  of  primary  disturbance  is  the  seat  of  a  chronic 
and  destructive  ulceration  from  which  the  disease  spreads  to  nu- 
merous secondary  foci  chiefly  by  lymphatic  metastasis.  Hektoen  and 
Perkins  describe  the  appearance  of  the  primary  lesion  in  Perkins' 
case  of  infection  by  S .  schenckii,  thus :  ' '  the  finger  from  the  first  to  the 
third  joints  is  swollen  to  twice  its  original  size,  presenting  in  the  center 
a  deep,  well-defined,  sharp,  undermined  ulceration,  the  size  of  a 
ten-cent  piece.  The  base  of  the  ulceration  is  rough  and  covered  with 
grayish-looking  pus.  This,  when  sponged  away,  leaves  a  bright  red 
surface;  the  ulcer  extends  through  the  whole  thickness  of  the  skin. 
Surrounding  the  ulcer  over  about  one-half  of  the  infiltrated  area,  are 
a  large  number  of  vesicles  and  a  few  pustules.  The  dorsal  surface 
of  the  hand  and  the  extensor  surface  of  the  forearm  present  a 
chain  of  swollen  lymphatics  along  which  are  about  twenty  nodules 
the  size  of  a  small  pea  to  a  large  hazel  nut.  .  .  .  This  little  patient 
does  not  complain  of  much  pain."  In  the  course  of  two  months 
Perkins  opened  and  treated  more  than  twenty  abscesses  resulting 
from  the  enlargement  and  softening  of  the  nodes. 

De  Beurmann  and  Gougerot  found  that  the  most  characteristic 
lesion  of  the  skin  is  a  nodule  in  which  three  processes  are  found,  some- 
times mixed  up  in  an  irregular  manner,  but  most  frequently  arranged 
concentrically.  "In  the  center  an  abscess  containing  polymorpho- 
nuclear  leukocytes  and  macrophages;  in  the  intermediate  zone  an 
area  of  degenerated  epithelioid  giant  cells  and  tuberculous  follicles, 
and  at  the  periphery  a  proliferation  of  basophile  lymph  and  con- 
nective-tissue cells  or  a  fibro-cellular  infiltration."  "The  structure 
of  the  sporotrichoma  is,  therefore,  very  closely  allied  to  that  of  the 
lesions  caused  by  syphilis,  tuberculosis,  and  by  the  agents  of  chronic 
suppuration,  and  it  resembles  sometimes  the  one,  sometimes  the 
other." 

De  Beurmann  and  Raymond,  1903,  and  de  Beurmann  and 
Gougerot,  1906,  describe  three  clinical  varieties  of  the  disease. 


764  Sporotrichosis 

1.  Disseminated  Gummatous  Sporotrichosis. — The  onset  is  insidious.     An 
accident  usually  leads  to  the  discovery  of  the  first  gummata.     The  number  of 
gummata  may  vary  up  to  100.     The  first  takes  origin  from  any  point  in  the  sub- 
cutaneous tissue.     Others  disseminate  themselves  over  the  whole  body.     Each 
gumma  has  an  autonomous  evolution  which  is  the  same  for  all.     At  first  it  is  a 
little  rounded  mass,  hard,  elastic,  painless  and  invariably  in  the  subcutaneous 
tissue.     The  mass  evolves  rapidly  in  the  direction  of  softening  and  in  four  or 
six  weeks  terminates  in  a  characteristic  cold  abscess.     When  it  undergoes  lique- 
faction, it  contains  a  fluid  which  is  at  first  transparent,  viscid,  gummy,  and  with 
purulent  streaks  and  later  becomes  opaque,  thick  and  purulent.     It  does  not 
undergo  complete  softening,  and  when  it  becomes  fluctuating  we  find  a  central 
cup-shaped  depression  surrounded  by  a  firm  and  resisting  zone,  and  when  its 
contents  are  evacuated,  there  remains  round  the  empty  pocket  a  persistent  and 
indurated  ring. 

2.  Disseminated  Subcutaneous,  Gummatous  Sporotrichosis  with  Ulceration. 
— In  this  variety,  the  subcutaneous  gummata  after  having  passed  through  the 
phases  described  above,  become  hypodermo-dermic  and  destroy  the  skin  by 
ulceration  more  or  less  rapidly,  sometimes  in  twenty  days,  sometimes  in  two  or 
three  months.     As  a  rule  the  ulcers  are  tuberculous  in  appearance.     Frequently 
the  ulceration  is  at  first  no  more  than  a  narrow  fistula  from  which  oozes  a  viscid, 
colorless  and  sometimes  reddish  pus  or  a  yellowish  serous  fluid. 

3.  Mixed    forms  are    frequent.     When    the  disease  has  existed  for  a  long 
time  it  presents  a  complete  clinical  picture.     Side  by  side  are  lesions  of  different 
age  with  different  tendencies  and  different  appearances;  tuberculous  looking, 
syphilitic  looking,  ecthymous,  rupial  and  furuncular.     There  may  be  associated 
lesions  of  thelymphatics,  and  lesions  of  the  dermis,  epidermis,  mucous  membranes, 
muscles,  osseous  tissues,  synovial  membranes,  eyes,  epididymis,  etc. 

4.  Localized   Sporotrichosis. — The  sporotrichum  penetrates  by  a  cutaneous 
lesion  at  the  site  of  which  it  produces  an  initial  lesion,  which  may  be  called  the 
"  sporotrichotic  chancre."     Then  it  gradually  invades  the  lymphatics  and  a  hard 
lymphatic  cord  studded  with  gummata — centripetal  gummatous  Sporotrichosis — 
makes  its  appearance.     Sometimes  the  lymph-nodes  of  the  region  react,  but  this 
is  not  constant.     The  disease  remains  localized  to  the  region  primarily  affected. 

Sporotrichosis  of  the  mucous  membranes,  of  the  muscles,  of  the  bones  and 
joints,  of  the  synovial  membranes,  of  the  eye,  of  the  epididymis,  of  the  kidney, 
and  of  the  lung  are  described  by  de  Beurmann.* 

Bacteriologic  Diagnosis. — Diagnosis  by  immediate  and  direct 
examination  of  the  pus  either  stained  or  unstained  is  difficult  be- 
cause the  parasites  are  few  in  number,  and  are  present  in  the  bacil- 
lary  form  that  is  so  difficult  to  recognize. 

The  approved  method  is  to  carefully  cleanse  the  skin  over  one 
of  the  closed  lesions,  disinfect  it  with  iodine,  and  then  puncture  the 
abscess  with  a  hollow  needle.  The  pus  obtained  is  spread  plenti- 
fully over  the  surface  of  culture-media  in  a  number  of  tubes  and  stood 
in  the  incubating  oven.  The  characteristic  colonies  should  appear  in 
from  four  to  twelve  days. 

Should  cultures  be  on  hand  in  the  laboratory  at  the  time  a  case 
presents  itself  for  diagnosis,  two  other  methods  may  be  employed. 

1.  The  Agglutination  Test. — A  suspension  of  the  spores  from  cul- 
tures of  the  sporotrichum  will  be  agglutinated  by  the   patient's 
serum  in  dilutions  of  1-400  to  1-500  on  the  average. 

2.  The  Complement-fixation  Test. — The  entire  culture  is  used  as 
an  antigen,  the  serum  of  the  patient  and  guinea-pig  complement  em- 
ployed as  usual.     As,  however,  oi'dium,  actinomyces,  discomyces 

*  "Brit.  Med.  Jour.,"  1912,  n,  293. 


Bacteriologic  Diagnosis  765 

and  other  fungi  give  the  same  degree  of  fixation,  the  method  lacks 
precision. 

Bloch  has  also  employed  an  intra-dermic  injection  of  a  sterilized 
emulsion  of  the  sporotrichum  for  purposes  of  diagnosis.  In 
twenty-four  hours,  patients  with  sporotrichosis  give  a  marked  re- 
action in  the  form  of  an  indurated  nodule  with  a  broad  reddish 
surrounding  areola. 


BIBLIOGRAPHIC  INDEX 


ABBOTT,  85, 156, 198,  207,  322,  586,  605 

Abbott  and  Bergey,  587,  588 

Abbott  and  Gildersleeve,  411,  693 

Abbott  and  Welch,  413 

Abderhalden,  140,  143 

Abderhalden  and  Freund,  142 

Abel,  no,  460,  548 

Abel  and  Claussen,  574 

Abel  and  Loffler,  594,  602,  621 

Abelous,  324 

Achard  and  Bensaude,  614 

Adami,  66,  73 

Adami  and  Chapin,  608 

Afanassiew,  441 

Agnew,  23 

Agramonte,  Reed,  and  Carroll,  538 

Agramonte,  Reed,  Carroll,  and  Lazear, 

536 
Alav,  438 

Albrech  and  Ghon,  391 
Albrecht,  552 
Alessi,  84 
Alt,  408 
Altmann,  223 
Alvarez  and  Tavel,  692 
Anaximander,  17 
Anderson  and  Forst,  384 
Anderson  and  Goldberger,  541,  542 
Anderson    and    McClintic,    253,    255, 

256,  257,  258,  260,  261 
Anderson  and  Rosenau,  103,  104,  132 
Andrevves  and  Gordon,  299 
Andrewes  and  Horder,  314 
Andrews,  175 
Anjeszky,  157 
Aoyama,  546 
Aristotle,  17 

Arloing,  96,  360,  683,  686 
Arnaud,  631 
Arning,  702 
Arnold,  171 
Arrhenius,  24 
Arustamoff,  37 
Aschoff,  no 

Aschoff  and  Gaylord,  166 
Audanard  and  Eberth,  324 
Auld,  448 
Austin,  517 
Axenfeld,  387,  408,  409,  452 

BABES,  313,  324,  372,  429,  433,  434, 

467,  710,  714 
Babes  and  Cornil,  433,  572 
Babes  and  Lepp,  98,  380 
Babes  and  Proca,  684 
Bacot,  554,  560,  561,  562 


767 


Bacot  and  Martin,  554 

Bail,  119,  123 

Baker,  507 

Baldwin  and  Trudeau,  680,  681 

Baldwin,  Graham,  and  Stewart,  337, 

339 

Banti,  212,  452 

Barbagallo  and  Casagrandi,  654 
Barker,  324 
Barker  and  Flint,  557 
Barker  (L.  F.),  324 
Barlow,  655 
Barren,  507 
Bass,  474,  484 
Bass  and  Johns,  484,  485 
Bassett  and  Duval,  648 
Bassett-Smith,  294,  468,  469 
Bateman,  513 
Baumgarten,   74,  432,  656,  670,  672, 

711 

Baumgarten  and  Walz,  682 
Bauzhaf  and  Steinhardt,  130 
Bayon,  510,  529 

Beattie  and  Dickson,  503,  504,  527 
Beck  and  Pfeiffer,  465 
Beck  and  Proskauer,  51,  667 
Becker,  307 
Beckman,  608 

Beebe,  Park,  and  Biggs,  424 
Behrend,  438 
Behring,  24,98,  127,  128,  129,  177,345, 

426,  456,  670,  676,  683,  689 
Behring  and  Kitasato,  99,  131 
Behring  and  Nissen,  109 
Behring  and  Nocht,  177 
Beitzke,  437 

Belfanti  and  Carbone,  116,  133 
Beninde,  669 

Bensaude  and  Achard,  614 
Benzanfon,  Griffon,  and  Le  Sours,  404 
Berestneff,  38 
Berestnew,  733 
Berg,  438 

Bergell  and  Meyer,  603 
Bergey,  587 

Bergey  and  Abbott,  587,  588 
Bergholm,  71 
Berkefeld,  174 
Bernheim,  683 

Bernheim  and  Hildebrand,  69 
Bernheim  and  Popischell,  437 
Bertarelli,  720 
Bertarelli  and  Bocchia,  175 
Bertarelli  and  Volpino,  721 
Bertrand  and  Phisalix,  99,  100,  132 
Besredka,  315,  387,  443,  558,  594 


768 


Bibliographic  Index 


Besredka  and  Metcchnikoff ,  268 
Besredka  and  Steinhardt,  104 
Besson,  333,  416,  556 
Bettencourt  and  Franca,  392 
Beyer,  Rosenau,  Parker,  and  Francis, 

539 

Beyerinck,  64 
Bezancon,  321 
Bielonovsky,  550 
Biggs,  423 

Biggs,  Park,  and  Beebe,  424 
Bignami,  472 
Billet,  478,  479 
Billroth,  23,  35,  233 
Binger  and  Wolbach,  515,  516 
Biondi,  70,  165 
Biondi  and  Heidenhain,  165 
Birch-Hirschfeld,  669 
Birt  and  Lamb,  468 
Bitter,  60,  575 
Bittu  and  Klemperer,  692 
Blacklock,  521 

Blaizot,  Nicolle,  and  Conseil,  498 
Blanchard,  25,  482,  492 
Blasi  and  Russo-Travali,  420 
Bloch,  765 
Blum,  324 
Blumer,  324 
Bockhart,  72,  306 
Boehm,  22 
Boland,  323 
Bellinger,  37,  732 
Bolton,  no,  138 
Bolton  and  Globig,  197 
Bolton  and  Pease,  53 
Bolton,  Dorset,  and  McBryde,  626 
Bomstein,  423 
Bonhoff,  588 

Bonney  and  Foulerton,  452 
Bonome,  98,  710 
Bonome  and  Gros,  54 
Bonome  and  Viola,  53 
Bordet,   24,   101,   116,  118,  120,   133, 

135,  139,  283,  718 
Bordet  and  Gay,  137 
Bordet  and  Gengou,  100,  280,  294,  441, 

442,  443 
Bordoni-Uffreduzzi,  327,  448,  452,  697, 

698 

Borrel,  556,  559 
Borrel  and  Roux,  350 
Bostrom,  732,  738 
Botkin,  217 
Bousfield,  498 
Bowhill,  549 
Boxmeyer,    McClintock,    and    Siffer, 

627 

Boyce,  741 

Boyce  and  Surveyor,  742,  744 
Brault,  507 
Braum,  652,  653 
Brebeck-Fischer,  438 
Brefeld,  40,  42 
Breinl,  498 
Brieger,  344,  575,  594 


Brieger  and  Cohn,  345 
Brieger  and  Ehrlich,  108,  332 
Brieger  and  Frankel,  76,  345,  357,  417, 

575,  593 
Brown,  400,  401,  404,  405,  412,  532, 

736 

Brown  and  Wright,  733,  734 
Bruce,  467,  469,  512,  513 
Bruce  and  Nabarro,  509,  513 
Bruce,  Nabarro,  and  Greig,  513 
Bruck  and  Neisser,  727 
Bruck  and  Wassermann,  726 
Bruck,  Wassermann,  and  Neisser,  279, 

281,  283 

Bruckner  and  Galasesco,  723 
Brues  and  Rosenau,  384 
Brumpt,  491,  493,  504,  507,  512,  513, 

517,  521,  653,  654 
Brunner,  629 

Buchner,  32,  108,  109,  135,  217,  219 
Buchner  and  Daremberg,  116 
Buerger,  447,  454 
Bujwid,  130 

Bullock  and  Hunter,  323 
Bumm,  306,  394 
Bumm  and  Nisot,  419 
Burn,  523 
Burri,  721 

Burroughs  and  McCollum,  428 
Burse,  39 
Busse,  747 

Buswell  and  Kraus,  602 
Biitschli,  472 
Buxton,  613,  614 
Buxton  and  Coleman,  611 
Buxton  and  Torry,  106 
Buxton  and  Vaughan,  124" 

CABOT,  378 

Cadio,  690 

Calkins,  27,  363,  364,  655 

Calkins  and  Williams,  636 

Calmette,  99,  132,  133,  324,  556,  559, 

604,  679 

Calmette  and  Gue"rin,  689 
Cameron,  672 
Camus  and  Gley,  133 
Canon,  462 

Canon  and  Pfeiffer,  24 
Cantani,  575 
Cantanni  (A.  Jr.),  465 
Capaldi,  610 

Carbone  and  Belfanti,  116,  133 
Cardan,  17 
Carmona  y  Valle,  536 
Carrasquilla,  704 
Carroll,  217,  537 
Carroll  and  Reed,  539 
Carroll,  Reed,  and  Agramonte,  538 
Carroll,  Reed,  Lazear,  and  Agramonte, 

536 

Carter,  536,  537,  742 
Carter  and  Hughes,  456 
Casagrandi  and  Barbagallo,  654 
Castellani,  25,  508,  729,  730,  731*  759 


Bibliographic  Index 


769 


azeneuve,  490 

jlli,  472,  478,  647 

jlli  and  Fiocca,  631,  633 

illi  and  Marchiafava,  386 

illi  and  Shiga,  615 

intanni  and  Tizzoni,  683 
[Chagas,  518,  520,  521,  523 
Chamberland,  173,  174,  360 
Chamberland  and  Roux,  98,  332 
Chamberland,  Pasteur,  and  Roux,  363 
Chantemesse,  590,  602,  604,  694 
Chantemesse  and  Widal,  599,  601,  602, 

647 

Chapin  and  Adami,  608 
Charm,  53 

Charrin,  98,  321,  694 
Charrin  and  Roger,  122,  85 
Chauffard  and  Quenu,  350 
Chauveau,  98,  105,  360 
Cheinisse,  403 
Chenot  and  Picq,  714 
Chester,  27,  36,  230 
Chevreul  and  Pasteur,  20 
Cheyne,  82 
Chisives,  92 
Christmas,  397,  398 
Christy,  Button,  and  Todd,  509 
Cienkowsky,  633 
Citron  and  Wassermann,  288 
Ciuffo,  726 
Clark,  431 

Clark  and  Flexner,  385 
Clark  and  Howard,  384 
Clarke  and  Miller,  175 
Claudius,  175 
Claussen  and  Abel,  574 
Clegg,  699,  701 

Clegg  and  Musgrave,  636,  637,  642,  644 
Cobbett,  89,  no,  431 
Cohn,  32,  294,  441 
Cohn  and  Brieger,  345 
Cohnheim,  656 
Colbach,  21 

Coleman  and  Buxton,  6n 
Coley,  54,  3 16 
Colla,  86 

Comte  and  Nicolle,  494,  531 
Comus  and  Gley,  138 
Conn,  80 
Conradi,  357 

Conradi  and  Drigalski,  608 
Conseil  and  Nicolle,  542 
Conseil,  Nicolle  and  Blaizot,  498 
Conseil,  Nicolle,  and  Couer,  541,  542 
Cooley  and  Vaughan,  619 
Cooley,  Vaughan,  and  Gelston,  75 
Coplin,  151 
Cornet,  657 
Cornevin,  332 
Cornevin  and  Thomas,  96 
Cornil  and  Babes,  433,  571 
Couer,  Nicolle,  and  Conseil,  541,  542 
Councilman,  309,  386,  431 
Councilman  and  Lafleur,  25,  632,  633, 
634 
49 


Councilman,  Mallory,  and  Pearce,  423 

Countess  del  Cinch6n,  472 

Courmont,  683 

Cousland,  730 

Coventon  and  Pelletier,  472 

Cowie,  692 

Cragg  and  Patton,  489,  501,  502,  517, 

.SiQ,  523,  562 
Craig,  635,  636,  639,  641,  642,  643,  644, 

655 

Crocker,  755 
Crooke,  313 
Crookshank,  739 
Cruveilheir,  131 
Cullum,  540 
Cumston,  621 
Cunningham,  533 
Curry,  460 

Curtis,  39,  343,  356,  357,  459,  667,  737 
Gushing,  596,  598,  614,  615 
Czaplewski,  692,  696,  697 
Czaplewski  and  Hensel,  441 
Czenzynke,  462 
Czerny,  316 

D ALTON  and  Eyre,  467 

Daniels,  163 

Danysz,  630 

Daremberg  and  Buchner,  116 

Darling,  534,  535,  636 

d'Arsonval,  53 

Davaine,  22,  353 

Davaine  and  Pollender,  24 

Davidson,  471 

Davis,  403,  404,  405,  441,  464 

Dean,  596 

de  Beurmann,  759,  761,  762,  763,  764 

de  Beurmann  and  Gougerot,  763 

de  Beurmann  and  Raymond,  763 

De  Foe,  543 

de  Geer,  505 

Deichler,  441 

Delage,  27 

Del6pine,  178 

Delezene,  102,  134 

Delius  and  Kolle,  465 

de  Mondeville,  21 

Denecke,  583,  588 

Denny,  412,  423 

Denys,  680 

Denys  and  Van  de  Veld*1,  307 

De  Renzi,  456 

de  Silvestri,  631 

De  Schweinitz,  627,  675,  684 

De  Schweinitz  and  Dorset,  626 

De  Schweinitz  and  Veasy,  408 

Descos  and  Nicholas,  73 

Detre,  283 

Detweiler,  75 

Deutsch,  97 

Deutsch  and  Feistmantel,  97 

Devell,  551 

Devonshire,  565 

Deycke,  197,  631 

Dickson  and  Beattie,  503,  504,  527 


770 


Bibliographic  Index 


di  Vestea  and  Maffucci,  684 

Dineur,  123 

Dobbin,  337 

Doderlein,  440 

Doderlein  and  Winterintz,  71 

Doflein,  328,  502 

Doleris  and  Pasteur,  308 

Dominici  and  Duval,  763 

Donitz,  in,  345,  350 

Donn6,  728 

Donovan,  525 

Donovan  and  Leishman,  25 

Donovan  and  Patton,  530 

Dopter  and  Vaillard,  651 

Dorser,  665 

Dorset,  665,  666 

Dorset  and  De  Schweinitz,  626 

Dorset,  Bolton,  and  McBryde,  626 

Dorset,  McBryde,  and  Niles,  626 

Douglas  and  Distaso,  30 

Douglas  and  Wright,  107,  270,  277 

Doutrelepont,  692 

Draper,  619 

Dreyfus,  620 

Drigalski  and  Conradi,  608 

Droba,  595 

Drysdale,  512 

Dubarre  and  Terre,  691 

du  Bary,  43 

Dubois,  416 

Du  Bois-Reymond,  53 

Duboscq  and  Leger,  653 

Ducrey,  403,  699 

Dujardin  and  Ehrenberg,  26 

Dunbar,  588 

Diingern,  100,  102,  116,  134 

Dunham,  199,  200,  333,  337,  414,  615, 

618,  622 

Dunham  and  Park,  648 
Durham,  116,  614 
Durham  and  Gruber,  122 
Durme,  305 
Dutton,  495,  506,  507 
Dutton  and  Forde,  25,  507,  509,  514 
Dutton  and  Todd,  494/495,  499,  507 
Dutton,  Todd,  and  Christy,  509 
Duval,  696,  699,  700,  701 
Duval  and  Bassett,  648 
Duval  and  Dominici,  763 
Duval  and  Vedder,  648 


EAGER,  544 

Eberth,  24,  241,  589,  615,  694 

Eberth  and  Audanard,  324 

Effront,  58 

Ehlers,  324 

Ehrenberg,  19,  240,  241 

Ehrenberg.  and  Diijardin,  26 

Ehrlich,  24,  91,  99,  100,  no,  in,  112, 
113,  118,  119,  123,  125,  129,  130, 
139,  152,  153,  154,  156,  158,  345, 
417,  427,  658,  659,  661,  663,  696 

Ehrlich  and  Brieger,  108,  332 

Ehrlich  and  Marshall,  121 


Ehrlich  and  Morgenroth,  101, 103,  no, 

i33»  !35»  283 

Eichhorn  and  Mohler,  709,  713,  714 
Eisenberg,  123,  230,  240,  241 
Eisner,  604 

Eisner  and  Wolff,  680,  726 
Elders,  433 
Ellermann,  434 
Elmassian  and  Morax,  422 
Elsching,  740 
Elser,  389 

Elser  and  Huntoon,  393 
Eisner,  605,  606,  622 
Emery,  ^413 
Emmerich,  616 
Emmerich  and  Low,  65,  323 
Emory,  70 
Empedocles,  17 
Endo,  610 

Engle  and  Reichel,  369 
Eppinger,  38 

Ernst,  130,  321,  324,  337,  338 
Ernst  and  Robey,  123 
Escherich,  33,  241,  615,  616 
Esmarch,  186,  197,  204,  207,  215,  238 
Evans,  182,  748 
Evans  and  Russell,  182 
Eyre,  44 
Eyre  and  Dal  ton,  467 


FANTHAM  and  Stephens,  506,  509 

Farranf  342 

Fasching,  457 

Favre,  726 

Fehleisen,  308,  318 

Fehling,  200 

Feistmantel  and  Deutsch,  97 

Feletti  and  Grassi,  471,  478,  479,  482 

Fermi,  60 

Fermi  and  Pernoss,  345 

Ferran,  579 

Fick,  432 

Field,  344 

Finkelstein,  324 

Finkler  and  Prior,  580,  581,  588 

Finlay,  536,  537 

Fiocca,  157,  647 

Fiocca  and  Celli,  631,  633 

Firth,  531,  533 

Fisch,  684 

Fischel  and  Wunschheim,  no 

Fischer  (E.),  113 

Fish,  1 20 

Fitzpatrick,  559 

Flatten,  386 

Flexner,  38,  312,  328,  333,  335,  387, 

390,  392,  393,  423,  632,  647,  650, 

651,  721 

Flexner  and  Clark,  385 
Flexner  and  Harris,  599 
Flexner  and  Jobling,  393 
Flexner  and  Lewis,  381 
Flexner  and  Noguchi,  25, 132, 133,  344> 

383,  384 


Bibliographic  Index 


771 


Flexner  and  Shiga,  651 

Flexner  and  Welch,  332,  337,  419 

Flint  and  Barker,  557 

Flournoy,   Norris  and  Pappenheimer, 

495,  497 
Fliigge,  55,  79,  107,  109,  151,  180,  181, 

240,  241,  243,  314,  321,  325,  340, 

351,  386,  445,  566,  669 
Fliigge  and  Hiss,  179 
Foa,  452 
Fodor,  107 

Foley  and  Sergent,  498 
Forde,  507 

Forde  and  Button,  25,  507,  509,  514 
Forneaca,  321 
Forssner,  120 
Forst  and  Anderson,  384 
Foulerton,  37 

Foulerton  and  Bonney,  452 
Fournier  and  Gilbert,  625 
Fox  and  Longcope,  445 
Fracastorius,  21 
Franca  and  Bettencourt,  392 
Francis  and  Grubs,  62 
Francis,  Rosenau,  Parker,  and  Beyer, 

539 

Frank  and  Heiman,  143 
Franke,  432 
Frankel,  55,  89,  215,  216,  226,  243,  244, 

332,  358,  362,  387,  388,  432,  443, 

444,  452,  457,  495,  571,  574,  575, 

576,  582,  584,  591,  599 
Frankel  and  Brieger,  76,  345,  357,  593 
Frankel  and   Pfeiffer,   304,   309,  330, 

34i,  342,  352,  354,  355,  4i5,  453, 

565,  570,  581,  586,  657,  668,  707 
Frankel  and  Trendenburg,  313 
Frankel  and  Weichselbaum,  328,  458 
Frankel  and  Wollstein,  443 
Frankforter,  182 
Frankland,  240,  241 
Fredericq,  107 

Freejmuth  and  Petruschky,  437 
Freire,  536 

Freund  and  Abderhalden,  143 
Freymuth,  579 
Friedlander,  31,   152,  410,   457,  459, 

460,  650 

Frisch,  324,  715,  716 
Frosch,  419 
Frosch  and  Kolle,  314 
Frost,  55,  208,  209,  210,  211,  238,  239 
Frothingham,  370 
Frothingham,  Page,  and  Paige,  762 
Frugoni,  667 
Fulleborn,  500 
Fuller,  1 88,  242 
Funck,  102 

GABBET,  661,  696 

Gabbi,  452 

Gaffky,  318,  319,  589,  599,  633 

Gaffky  and  Koch,  632 

Galasesco  and  Bruckner,  723 

Galeotti,  61 


Galli-Valerio,  54,  553,  631 

Galtier,  363 

Gamaleia,  445,  450,  575,  584,  586,  588 

Garini,  200 

Garr6,  72,  306 

Gartner,  614,  615,  624,  650 

Gaspard,  20 

Gaultier  de  Sauvage,  540 

Gauss  and  Schumburg,  31 

Gauthier  and  Jodasshon,  726 

Gaveno  and  Girard,  541 

Gay,  104 

Gay  and  Bordet,  137 

Gay  and  Southard,  104 

Gaylord  and  AschofT,  166 

Geddings  and  Wasdin,  536 

Gels  ton,  75 

Gelston  and  Marshall,  75 

Gelston,  Vaughan,  and  Cooley,  75 

Gengou  and  Bordet,  100,  280,  294,  441, 

442,  443 
Gerhard,  540 

.  Germano  and  Maurea,  599 
Gescheidel  and  Traube,  135 
Gessard,  61,  240,  321 
Gheorghiewski,  100,  323 
Ghon,  550,  552 
Ghon  and  Albrech,  391 
Ghoreyeb,  719 
Ghriskey  and  Robb,  69,  300 
Gibier,  85 
Gibson,  130 

Giemsa,  163,  365,  366,  368,  384,  726 
Giemson,  368 
Gilbert  and  Fournier,  625 
Gilbert  and  Roger,  690 
Gilbert,  Zinsser,  and  Hopkins,  724 
Gilchrist,  39,  747,  748,  751 
Gilchrist  and  Stokes,  747,  749 
Gildersleeve  and  Abbott,  411,  693 
Gilliland  and  Pearson,  689 
Girard  and  Gaveno,  541 
Gley  and  Camus,  133 
Gley  and  Comus,  138 
Globig  and  Bolton,  197 
Goldberger  and  Anderson,  541,  542 
Goldhorn,  719 
Goldschmidt,  389 
Golgi,  472,  474,  478,  479 
Gomez,  472 
Goodby,  70 
Goodsir,  233 

Goodwin  and  Sholly,  392 
Gb'ppert,  386 
Gorden,  160 

Gordon,  314,  315,  546,  577 
Gordon  and  Andrewes,  299 
Gorgas,  539 
Gorham,  62 

Gottschalk  and  Immerwahr,  71 
Gottstein,  103 

Gougerot  and  de  Beurmann,  763 
Gourvitsch,  653 
Gradenigo,  324 
Graham  and  Irons,  38,  750 


772 


Bibliographic  Index 


Graham,  Stewart,  and  Baldwin,  337, 

339 

Graham-Smith,  498 

Gram,  152,  153, 154, 165,  300,  302,  308, 
310,  318,  319,  321,  322,  325,  329, 
332,  334,  340,  35i,  352,  354,  384.- 
385,  388,  389,  393,  394,  395,  398, 
400,  401,  403,  406,  408,  409,  411, 
413,  444,  457,  462,  467,  494,  497, 
543,  546,  564,  566,  568,  584,  589, 
590,  624,  625,  626,  627,  628,  629, 
649,  656,  659,  663,  694,  695,  696, 
706,  707,  715,  718,  732,  734,  741, 

743,  745,  761 
Gram  and  Weigert,  154 
Grassi,  475 

Grassi  and  Feletti,  471,  478,  479,  482 
Grawitz,  39,  438,  440 
Greig,  Bruce,  and  Nabarro,  513 
Greite,  133 

Griffon,  Benzangon,  and  Le  Sours,  404 
Grigorjeff,  631 
Grigorjeff  and  Ukke,  332 
Grimme,  146 
Grixoni,  342 
Grohman,  107 
Gromakowsky,  316 
Gros  and  Bonome,  54 
Grosset,  440 
Gruber,  116,  215 
Gruber  and  Durham,  122 
Gruber  and  Wiener,  578 
Gr  ubler,  146,  155 
Grubs  and  Francis,  62 
Gruby,  438,  752 
Griinbaum,  599 
Griinbaum  and  Widal,  603 
Grysez  and  van  Steenberghe,  73 
Gscheidel  and  Traube,  107 
Guarniere,  448 
Guerin  and  Calmette,  689 
Guiart,  516 
Guidi,  438 
Guiteras,  539 
Giinther,  193,  234,  238,  243,  302,  331, 

588 

Giinther  and  Wagner,  721 
Gwyn,  598,  614 

HAECKEL,  26 

Haffkine,  97,  263,  546,  547,  557,  558, 

578,  579,  600 
Halberstadter,  731 
Hall,  527 
Hallein,  438 
Hallier,  233 
Hamburger,  604 
Hamerton,  513 
Hamilton,  431 
Hankin;  55,  85,  108,  361 
Hankin  and  Leumann,  549 
Hankin  and  Wesbrook,  357 
Hansen,  24,  58,  216,  440,  695,  697 
Harris,  368,  379,  646 
Harris  and  Flexner,  599 


Harris  and  Shackell,  378 

Hartmann,  636 

Harvey,  118 

Harvey  and  McKendrick,  380 

Hashimoto,  574 

Hasslauer,  72 

Haupt,  409 

Hauser,  325,  326 

Havelburg,  536,  555 

Hebra,  752 

Heidenhain,  165 

Heidenhain  and  Biondi,  165 

Heider,  588 

Heim,  303,  320,  438,  463,  572,  591,  617 

Heiman,  396 

Heiman  and  Frank,  143 

Heinemann,  359 

Hektoen,  309 

Hektoen  and  Perkins,  759,  760,    761, 

762,  763 
Henle,  22 

Hensel  and  Czaplewski,  441 
Herman,  82 
Herzog,  553 

Hesse,  219,  234,^235,  610 
Hesse  and  Liborius,  219 
Hewlett,  in,  113,  614 
Hewlett  and  Nolen,  424 
Heymans,  in 
Heyman-Sticher,  669 
Hildebrand,  roo 
Hildebrand  and  Bernheim,  69 
Hill,  145,  146,  207,  253,  609 
Hippo.crates,  631 
Hirsh,  312 
Hiss,  44,  318,  390,  446,  448,  455,  592, 

607,  622,  679,  721 
Hiss  and  Fliigge,  179 
Hiss  and  Russell,  648 
Hiss  and  Zinsser,  36,  188,  310,  319,  387, 

396,  447,  448,  456,  589,  614,  708, 

749 

Hodenpyl,  671 
Hodenpyl  and  Prudden,  686 
Hoffa,  357 
Hoffmann,  495 
Hoffmann  and  Muhlens,  723 
Hoffmann  and  Prowazek,  728 
Hoffmann  and  Schaudinn,  24,  69,  718, 

728,  730 

Hofmann,  423,  429,  430,  431,  592 
Hogyes,  374,  378,  385 
Holmes,  22 
Hoist,  80,  314 

Hopkins,  Zinsser,  and  Gilbert,  724 
Horder,  464 

Horder  and  Andrewes,  314 
Howard,  420,  429,  431,  452,  460,  487 
Howard  and  Clark,  384 
Howard  and  Perkins,  317 
Howard,  Jr.,  339 
Hiickel  and  Losch,  42 
Hughes,  469 
Hughes  and  Carter,  456 
Humer,  595 


Bibliographic  Index 


773 


Huntemiiller  and  Lentz,  383 
Hunter  and  Bullock,  323 
Huntoon  and  Elser,  393 
Hunziker,  215 

Hiippe,  241,  361,  566,  571,  650 
Hiippe  and  Wood,  362 
Huxley,  27 

INCHLEY  and  Nuttall,  122 

Irons,  349 

Irons  and  Graham,  38,  750 

Irons  and  Savage,  607 

Israel,  732,  734,  738,  740 

Issaeff,  122 

Issaeff  and  Kolle,  577 

Itzerott  and  Niemann,  321,  581,  583, 

58S,  694 
Iwanow,  357 

JACKSON,  534,  612 
Jacob,  619 

acobsohn  and  Pick,  388 

acoby,  120 

adkewitsch,  324 

ager,  241,  386,  387 

amieson  and  Johnston,  703 

asuhara  and  Ogata,  99,  361 
Jenner,  93,  95,  163,  263,  276 
Jez,  602 
Jobling  and  Flexner,  393 

ochmann  and  Krauss,  441 

odasshon  and  Gauthier,  726 

ohannsen  and  Riley,  489 
_ohns  and  Bass,  484,  485 
Johnson,  509,  614 
Johnston  and  Jamieson,  703 
Joos,  123 

Jordan,  241,  323,  602,  615 
Jordan,  Russel,  and  Zeit,  593 
Jorgensen,  58 
Justinian,  543 

KAENSCHE,  59 

Kamen,  348 

Kanthack,  742 

Kaplan,  290 

Kaposi  and  Kundrat,  755 

Karlinski,  299,  324 

Karlinski  and  Lubarsch,  624 

Kartulis,  328,  406,  632,  633,  634 

Kashida,  606 

Kastle,  Rosenau,  and  Lumsden,  594 

Kazarinow,  651 

Keen,  596 

Kehrer,  438 

Keidel,  281 

Kerr,  MacNeal,  and  Latzer,  71 

Kimla,  Poupe",  and  Vesley,  666 

Kinghorn  and  Todd,  498 

Kinghorn  and  Yorke,  513 

Kircher,  17,  21 

Kirchner,  400,  401 

Kitasato,  24,  98,  99,  174,  219,  226,  340, 
344,  347,  348,  446,  463,  543,  545, 
546,  547,  549,  559,  574,  575,  632 

Kitasato  and  Behring,  99,  131 


Kitasato  and  Weil,  219 

Kitasato  and  Yersin,  24 

Kitt,  96,  712 

Klebs,  23,  105,  411,  575,  631,  656,  675, 

680 

Klein,  548,  551,  554 
Klemperer,  452 
Klemperer  and  Bittu,  692 
Klemperer  and  Levy,  461,  592,  594 
Klemperer  (G.  and  F.),  455 
Klencki,  620 
Klimenko,  443,  654 
Kline,  332,  623 
Knapp,  43 1 

Knapp  and  Novy,  497,  500,  501 
Knapp,  Levaditi,  and  Novy,  497 
Knisl,  582 
Knopfelmacher,  381 
Knorr,  344,  556 
Kny,  41 
Koch,  22,  23,  24,  50,  106,  178,  196,  201, 

204,    205,    206,   207,    220,    222,    223, 

237,  252,  308,  324,  329,  352,  353, 
358,  359,  4o6,  407,  475,  485,  495, 
497,  499,  504,  514,  565,  568,  569, 
57o,  57.i,  574,  575,  576,  582,  585, 
588,  589,  633,  634,  656,  657,  658, 
660,  661,  664,  665,  670,  671,  676, 
677,  680,  681,  682,  683,  685,  686, 
687,  725 

Koch  and  Gaffky,  632 

Kock  (C.  L.),  501 

Kohlbrugge,  71 

Kolisko  and  Paltauf,  419 

Kolle,  151,  555,  556 

Kolle  and  Delius,  465 

Kolle  and  Frosch,  314 

Kolle  and  Issaeff,  577 

Kolle  and  Otto,  307,  558 

Kolle  and  Pfeiffer,  594,  600,  602 

Kolle  and  Strong,  558 

Kolle  and  Wassermann,  37, 40, 42,  no, 
392,  393,  439,  48o,  482,  483,  495, 
496,  547,  696,  697,  757 

Kolmer,  141,224,  .81,388,389,428,429 

Koplik,  441 

Korn,  693 

Kossee  and  Overbeck,  550 

Kossel,  99,  100,  133,  138,  324 

Krai,  757 

Krannhals,  324 

Kratter,  396 

Kraus,  100,  116,  120,  398 

Kraus  and  Buswell,  602 

Kraus  and  Levaditi,  374 

Kraus  and  Wernicke,  383 

Krauss,  305 

Krauss  and  Jochmann,  441 

Krefting,  403 

Kronig,  175 

Kronig  and  Menge,  337 

Kronig  and  Paul,  177 

Kruger,  53 

Krumweide  and  Park,  687 

Krumweide  and  Pratt,  434 


774 


Bibliographic  Index 


Kruse,  79,  325,  351,  509,  566,  618,  632, 

648 

Kruse  and  Pasquale,  632 
Kubel  and  Tiemann,  608,  609 
Kiihne,  707 

Kundrat  and  Kaposi,  755 
Kurloff,  441 
Kurth,  311,  3 13 
Kutcher,  709 
Kutschbert  and  Neisser,  431 

LABBE,  478,  479,  482 

Laennec,  673 

Lafleur  and  Councilman,  25,  632,  633, 

634 

Laitinen,  396 

Lamar  and  Meltzer,  451,  460 
Lamb  and  Birt,  468 
Lambert,  345,  374,  453 
Lambert,  Steinhardt,  and  Poor,  365 
Lambl,  631,  633 
Lammershirt,  433 
Landois,  133 
Landsteiner,  102 
Landsteiner  and  Popper,  381 
Langenbeck,  438   732 
Laplace,  177 
Larkin,  38 

Lartigau,  320,  324,  674 
Laschtschenko,  669 
Lassar,  718 
Latapie,  135,  225,  226 
Latour,  19 

Latour  and  Schwann,  19 
Latzer,  MacNeal,  and  Kerr,  71 
Laurent,  438 
Laveran,  25,  471,  472,  474,  477,  478, 

479,  482,  494,  509 

Laveran  and  Mesnil,  507,  508,  510,  525 
La  Wall  and  Leffmann,  192 
Lazear,  538 
Lazear,  Reed,  Carroll,  and  Agramonte, 

536 

Leach,  75,  505 
Leber,  43,  305,  432 
Leclainche  and  Nocard,  482 
Le  Dantec,  340 
Ledderhose,  323 
Leeuwenhoek,  18,  19,  26 
Leffmann  and  La  Wall,  192 
Leger  and  Duboscq,  653 
Lehman  and  Neumann,  230,  232 
Leichtenstern,  386 
Leishman,  163,  270,  276,  277,  499,  525, 

526,  528 

Leishman  and  Donovan,  25 
Lemoine,  313 
Lenglet,  405 

Lennholm  and  Miller,  58 
Le  Noir,  324 
Lentz,  648 

Lentz  and  Huntemiiller,  383 
Leo,  85,  714 
Lepierre,  391 
Lepp  and  Babes,  98,  380 


Lesage,  240,  621 

Lesage  and  Thiercelin,  324 

Le  Sours,  Benzancon,  and  Griffon,  404 

Leubarth,  313 

Leuchs  and  von  Lingelsheim,  392 

Leumann   557 

Leumann  and  Hankin,  549 

Levaditi,  276,  721 

Levaditi  and  Kraus,  374 

Levaditi  and  Manouelian,  722 

Levaditi  and  Mclntosh,  719,  722 

Levaditi  and  Nattan-Larrier,  731 

Levaditi  and  Yamanouchi,  280 

Levaditi,  Novy,  and  Knapp,  497 

Levene,  676 

Levin,  315 

Levy,  452,  680 

Levy  and  Klemperer,  461,  592,  594 

Levy  and  Steinmetz,  709 

Lewis,  25 

Lewis  and  Flexner,  381 

Lexer,  358 

Leyden,  603 

Libman,  314,  614 

Liborius,  216,  219 

Liborius  and  Hesse,  219 

Lichtowitz,  433 

Liebig,  20 

Lignieres,  679 

Limbourg,  611 

Lincoln  and  McFarland,  456 

Lindemann,  102 

Lindt,  43 

Lingelsheim,  304,  310,  386 

Link,  759 

Linn,  505 

Linossier,  438 

Linton  and  Thomas,  509 

Lisbon,  553 

Lister,  23,  172 

Livingstone,  512 

Loag  and  Van  der  Pluyn,  394 

Lock  wood,  174 

Lofner,  24,  151,  158,  197,  206,  333,  351, 
389,  395,  408,  411,  413,  414,  415, 
416,  417,  429,  577,  611,  615,  628, 
629,  707,  710 

Lb'rfler  and  Abel,  594,  602,  621 

Loffler  and  Schiitz,  24,  566,  706,  711 

Longcope,  614 

Longcope  and  Fox,  445 

Lord,  400,  401 

Losch,  25,  631,  633,  634,  642 

Losener,  599 

Low,  522,  599 

Low  and  Emmerich,  65,  323 

Low  and  Sambon,  473 

Lowden  and  Williams,  364,  368,  370, 

37i 

Lubarsch,  109,  360 
Lubarsch  and  Karlinski,  624 
Lubarsch  and  Oestertag,  387 
Lubbert,  177 
Lubenau,  315 
Lubinski,  351 


Bibliographic  Index 


775 


Lugol,  152 

Ltihe,  477 

Lumsden,  Rosenau,  and  Kastle,  594 

Lutz,  763 

Lutz  and  Splendore,  759 

Luzzani,  369 

MACCALLUM,  473,  474,  477 

MacConkey,  612 

Macfadyen,  449,  594,  602 

Macfadyen  and  Rowland,  76,  594 

Macgregor,  631 

Mackie,  498,  513 

MacNeal,  Latzer,  and  Kerr,  71 

Madsen,  24,  102,  in,  131 

Madsen  and  Noguchi,  132 

Madsen  and  Salmonson,  115 

Maffucci  and  di  Vestea,  684 

Mafucci,  690 

Magendie,  103 

Maggiora,  68,  324,  631 

Maher,  619,  659 

Malassez,  694 

Mallory,  155,  368,  597,  647 

Mallory  and  Wright,  163, 165,  220,  386 

Mallory,  Councilman,  and  Pearce,  423 

Malmsten,  652,  752 

Malvoz,  123 

Manceaux  and  Nicolle,  533 

Mann,  369 

Mannatti,  305 

Manouelian  and  Levaditi,  722 

Manson,  413,  472,  473,  477,  488,  506, 

507,  529,  655 
Manuelian,  365 
Maragliano,  683 
Marburg,  294 
Marchiafava,  472 
Marchiafava  and  Celli,  386 
Marchoux,  361 
Marchoux  and  Salimbeni,  494 
Marie,  380 

Marie  and  Morax,  346 
Marino,  163,  164,  165,  276 
Marks,  383 
Marmier,  357 

Marmorek,  81,  314,  315,  316,  453,  456 
Marshall  and  Ehrlich,  121 
Marshall  and  Gelston,  75 
Martha,  324 

Martin,  127,  132,  323,  357 
Martin  and  Bacot,  554 
Martin  and  Meyer,  500 
Martin  and  Roux,  no 
Marx,  146,  377 
Masselin,  590 
Masselin  and  Thoinot,  564 
Masterman,  532 
Matruchat,  760 
Matschinsky   and    Rymowitsch,    407, 

409 

Matterstock,  692 
Mattson,  103 
Matzenauer,  433 
Maurea  and  Germano,  599 


Mayer,  351,  369 

McBryde,  Dorset,  and  Bolton,  626 
McBryde,  Dorset,  and  Niles,  626 
McCarthy  and  Ravenel,  372 
McClintic    and   Anderson,    253,    255, 

256,  257,  258,  260,  261 
McClintock,    Boxmeyer,    and    Siffer, 

627 

McCollum  and  Burroughs,  428 
McConkey,  650 
McConnell,  703 
McCoy  and  Smith,  551 
McDaniel  and  Wilson,  424 
McFadyen,  689,  709 
McFarland,  362,  427,  684 
McFarland  and  1'Engle,  277 
McFarland  and  Lincoln,  456 
McFarland  and  Small,  62 
Mclntosh  and  Levaditi,  719,  722 
Mclntyre,  75 

McKendrick  and  Harvey,  380 
McNeal,  527 

McNeal  and  Novy,  510,  521,  700 
Megnin,  512 
Meier  and  Forges,  280 
Meirowsky,  726 
Melcher  and  Ortmann,  701 
Meltzer  and  Lamar,  451,  460 
Menge  and  Krb'nig,  337 
Mense,  526 
Mesnil,  107,  509,  530 
Mesnil  andLaveran,  507,  508,  510,  525 
Messea,  31 
Metalnikoff,  102,  134 
Metschnikoff,  24,  88,  90,  101,  102,  106, 

107,  108,  109,  no,  116,  118,  119, 

122,  123,  138,  270,  718 
Metschnikoff  and  Besredka,  268 
Metschnikoff  and  Roux,  718 
Meunier,  54 

Meyer,  149,  150,  222,  223 
Meyer  and  Bergell,  603 
Meyer  and  Martin,  500 
Meyer  and  Ransom,  346,  347 
Michel,  416 
Migula,  27,  32,  34,  35,  36,  230,  232, 

233,  445,  458«  616,  629,  658,  717 
Miller,  69,  70,  272,  273,  274,  275,  276, 

433,  494,  588,  595 
Miller  and  Clarke,  175 
Miller  and  Lennholm,  58 
Milne  and  Ross,  494,  495,  499 
Miquel,  235 
Mitchell,  22 

Mitchell  and  Stewart,  133 
Mithridates,  98 
Mittman,  68 
Moczutkowski,  540 
Moffit  and  Ophiils,  748 
Mohler  and  Eichhorn,  709,  713,  714 
Moller,  157,  499,  692,  693 
Monnier,  324 
Montague,  92 

Montesano  and  Montesson,  349 
Montesson  and  Montesano,  349 


776 


Bibliographic  Index 


Montgomery,  73,  748,  749,  751 

Montgomery  and  Walker,  748 

Monti,  450 

Moon,  366 

Moore  and  Taylor,  709 

Morax,  406,  407,  408,  409 

Morax  and  Elmassian,  422 

Morax  and  Marie,  346 

Morgenroth,  100,  in,  116,  121 

Morgenroth   and    Ehrlich,    101,    103, 

no,  133,  135,  283 
Mori,  458 
Moriya,  687 
Moro,  108,  679 
Morse,  305 
Moschcowitz,  349 
Moser,  316 
Moses,  695 
Mosso,  133 
Mott,  515 
Motz,  324 
Mouton,  107 
Much,  659 

Muhlens  and  Hoffmann,  723 
Muir  and  Ritchie,  153,  156,  160,  649, 

650 

Miiller,  150,  213,  582,  755 
Murchison,  540 
Murphy,  740 
Murray,  499,  502 
Musgrave  and   Clegg,   636,  637,  642, 

644 

Musgrave  and  Strong,  647,  648,  654 
Myers,  99,  101,  102,  in,  120 

NABARRO  and  Bruce,  509,  513 
Nabarro,  Bruce,  and  Greig,  513 
Nattan-Larrier  and  Levaditi,  73 1 
Nee'low,  74 
Negri,  363,  364,  365,  366,  367,  368,  369, 

.  370,  37i,  372 

Neisser,  24,  394,  401,  413,  588,  727 
Neisser  and  Bruck,  727 
Neisser  and  Kutschbert,  431 
Neisser  and  Sachs,  139 
Neisser  and  Wechsberg,  136,  137,  138, 

.  3°5,  307 
Neisser,  Wassermann,  and  Bruck,  279, 

281,  283 
Neiva,  524 

Nelis  and  Van  Gehuchten,  372 
Nepveu,  507 
Nessler,  64 
Netter,  452 
Neufeld,  454 

Neuman  and  Swithinbank,  246 
Neumann,  324,  424,  441 
Neumann  and  Lehman,  230,  232 
Newman,  161 

Newman  and  Swithinbank,  669 
Newmark,  294 
Newsholme,  595 
Nicati  and  Rietsch,  575,  576 
Nicholas,  726 
Nicholas  and  Descos,  73 


Nicholls,  73 

Nichols  and  Schmitter,  217,  218 

Nicolaier,  24,  340 

Nicolaysen,  397 

Nicolle,  154,  403,  433,  527,  530,  531, 

534,  541,  701 

Nicolle  and  Comte,  494,  531 
Nicolle  and  Conseil,  542 
Nicolle  and  Manceaux,  533 
Nicolle,  Blaizot,  and  Conseil,  498 
Nicolle,  Couer,  and  Conseil,  541,  542 
Niemann  and  Itzerott,  321,  581,  583, 

585,  694 

Niles,  Dorset,  and  McBryde,  626 
Nisot  and  Bumm,  419 
Nissen,  109,  178 
Nissen  and  Behring,  109 
Nitzsch,  505 
Noble,  726 

Nocard,  37,  348,  350,  614,  625,  665 
Nocard  and    Leclainche,  482 
Nocard  and  Railliet,  512 
Nocard  and  Roux,  195,  665 
Nocht  and  Behring,  177 
Noguchi,  25,  132,  133,  280,  282,  283, 

287,  294,  295,  296,  297,  365,  366, 

498,  719,  723,  724,  727,  728 
Noguchi  and  Flexner,  25,  132,  133,  344, 

383.,  384 

Noguchi  and  Madsen,  132 
Noissette,  440 
Nolen  and  Hewlett,  424 
Nolf,  120 
Norris,  38 

No  iris  and  Oliver,  61 
Norris,  Pappenheimer,  and  Flournoy, 

495,  497 
Novy,  148,  215,  216,  217,  229,  495,  496, 

497,  S27,  S31'  627,  692 
Novy  and  Knapp,  497,  500,  501 
Novy  and  McNeal,  510,  521,  700 
Novy  and  Vaughan,  58,  247 
Nuttall,  107,  108,  121,  122,  133,  146, 

147,  359,  496,  501,  552,  553,  662 
Nuttall  and  Tnchley,  122 
Nuttall  and  Welch,  332,  334,  337 

OBERMEIER,  23,  24,  494 

Oertel,  419 

Oestertag  and  Lubarsch,  387 

Oettinger,  324 

Ogata,  545,  549,  553,  631 

Ogata  and  Jasuhara,  99,  361 

Ogston,  302,  308 

Ohlmacher,  426,  429,  599,  622 

Oliver  and  Norris,  61 

Olsen,  438 

Ophuls,  39,  748 

Ophiils  and  Moffit,  748 

Oppenheim,  294 

Oriste-Armanni,  566 

Orlowski  and  Palmirski,  417 

Orth,  154 

Ortmann,  448 

Ortmann  and  Melcher,  701 


Bibliographic  Index 


777 


Oshida,  375 

Osier,  631,  632,  633 

Otero,  541 

Otto,  103 

Otto  and  Kolle,  307,  558 

Overbeck  and  Kossee,  550 

Ovid,  17 

Oviedo,  729 

PAGE,  Frothingham,  and  Paige,  762 

Paige,  Frothingham,  and  Page,  762 

Palmirski  and  Orloswki,  417 

Paltauf,  42 

Paltauf  and  Kolisko,  419 

Pane,  456 

Panfili,  177 

Pansini,  324 

Pappenheim,  661 

Pappenheimer,  Norris,  and  Flournoy, 

495,  497 
Paquin,  683 
Pariette,  609 
Park,  81,  220,  349,  388,  390,  413,  425, 

428,  429 

Park  and  Dunham,  648 
Park  and  Krumwiede,  687 
Park,  Biggs,  and  Beebe,  424 
Parke  and  Williams,  445 
Parker,  Rosenau,  Francis,  and  Beyer, 

539 

Pasquale  and  Kruse,  632 
Passet,  300,  308,  616 
Passler,  456 
Pasteur,  19,  20,  24,85,95,96,  105,  113, 

173,  215,  218,  263,  267,  329,  358, 

360,  361,  363,  373,  374,  377,  378, 

444,  564,  565 
Pasteur  and  Chevreul,  20 
Pasteur  and  Doleris,  308 
Pasteur  and  Toussaint,  564 
Pasteur,  Chamberland,  and  Roux,  363 
Paterson,  684 
Patton,  529,  530 
Patton  and  Cragg,  489,  501,  502,  517, 

519,  523,  562 
Patton  and  Donovan,  530 
Paul  and  Kronig,  177 
Paulicki,  690 
Pawlowski,  54,  666 
Pawlowsky,  578,  579 
Peabody  and  Pratt,  596,  604,  611 
Pearce,  313,  420,  421 
Pearce,  Councilman,  and  Mallory,  423 
Pearson,  710 

Pearson  and  Gilliland,  689 
Pease  and  Bolton,  53 
Peckham,  620 
Pelletier  and  Coventou,  472 
Perkins,  324,  459,  742,  763 
Perkins    and  Hektoen,  759,  760,  761, 

762,  763 

Perkins  and  Howard,  317 
Pernoss  and  Fermi,  345 
Perroncita,  615 
Perroncito,  564 


Peterson,  403 

Petkowitsch,  608 

Petri,  204,  206,  207,  217,  218,  235,  236, 

430,  693 
Petruschky,  36,  38,  199,  314,  598,  599, 

615,  624,  678 

Petruschky  and  Freejmuth,  437 
Pfeiffer,  101,  116,  135,  151,  195,  462, 

463,  464,  465,  466,  578,  584,  585, 

586,  587,  694 
Pfeiffer  and  Beck,  465 
Pfeiffer  arid  Canon,  24 
Pfeiffer  and   Frankel,  304,  309,  330, 

341,  342,  352,  354,  355,  4i5,  453, 

565,  570,  581,  586,  657,  668,  707 
Pfeiffer  and  Kolle,  594,  600,  602 
Pfeiffer  and  Vogedes,  578 
Pfuhl,  327,  676 

Phisalix  and  Bertrand,  99,  100,  132 
Piaget,  505 
Pianese,  530 
Piatkowski,  663 
Pick  and  Jacobsohn,  388 
Picq  and  Chenot,  714 
Pictet  and  Yung,  55 
Pierce,  452 
Piorkowski,  608,  622 
Pirquet,  427,  679,  726 
Pirquet  and  Schick,  103 
Pitfield,  159,  345,  627 
Platania,  85 

Plaut,  42,  433,  438,  439,  440,  757 
Plencig,  21 
Plett,  93 
Pohl,  241 
Pollender,  22,  353 
Pollender  and  Davaine,  24 
Poor  and  Steinharflt,  367 
Poor,  Steinhardt,  and  Lambert,  365 
Pope,  602 

Popischell  and  Bernheim,  437 
Popper  and  Landsteiner,  381 
Porges  and  Meier,  280 
Portier  and  Richet,  103 
Posadas  and  Wernicke,  748 
Pott,  54 

Poupe",  Kimla,  and  Vesley,  666 
Pratt  and  Krumweide,  434 
Pratt  and  Peabody,  596,  604,  611 
Preindelsberger,  69 
Prescott,  58,  616 
Prescott  and  Winslow,  199 
Prior  and  Finkler,  580,  581,  588 
Proca  and  Babes,  684 
Proskauer  and  Beck,  51,  667 
Proskauer  and  Voges,  650 
Prowazek,  328,  511 
Prowazek  and  Hoffmann,  728 
Prudden,  239,  312,  671 
Prudden  and  Hodenpyl,  686 

QUENU  and  Chauffard,  350 
Quincke,  755 
Quincke  and  Roos,  632 
Quinquaud,  438 


778 


Bibliographic  Index 


RABINOWITSCH,  246,  693,  747 

Railliet  and  Nocard,  512 

Ramon,  345 

Ranken,  511 

Ransom  and  Meyer,  346,  347 

Rappaport,  549 

Raskin,  313 

Ravenel,   55,  73,  194,  197,  198,  201, 

202,  215,  240,  241,  244,  686 
Ravenel  and  McCarthy,  372 
Raymond  and  de  Beurmann,  763 
Redi,  17,  18 
Reed  and  Carroll,  539 
Reed,  Carroll,  and  Agramonte,  538 
Reed,  Carroll,  Lazear,  and  Agramonte, 

536 

Reed,  Vaughan,  and  Shakespeare,  595 
Reich  el,  174,  666 
Reichel  and  Engle,  369 
Remak,  755 
Remy,  606 
Ress,  438 
Rettger,  71,  361 
Reyes,  416 
Ribbert,  305,  307 
Richards,  715,  716 
Richardson,  598,  605 
Richet  and  Portier,  103 
Ricketts,  748 
Ricketts  and  Wilder,  542 
Rideal,  178 

Rideal  and  Walker,  253 
Ridi,  505 

Riedel  and  Wolffhiigel,  570 
Rieder.  54 
Rieger,  386 
Rietsch,  575 

Rietsch  and  Nicati,  575,  576 
Riley  and  Johannsen,  489 
Rindfleisch,  585 
Rist,  418 
Ritchie,  350 

Ritchie  and  Muir,  153,156,160,649,650 
Ritter,  441 
Rivolta,  25,  690 
Robb  and  Ghriskey,  69,  300 
Robb  and  Welch,  174 
Robertson,  593 
Robey  and  Ernst,  123 
Robin,  438 
Robinson,  182 
Rodet,  307 

Roger,  82,  84,  361,  440,  694 
Roger  and  Charrin,  85,  122 
Roger  and  Gilbert,  690 
Rogers,  399,  525,  527,  529,  642 
Rogone,  175 
Rolleston,  619 
Roloff,  690 

Romanowsky,  163,  165,  367 
Roos  and  Quincke,  632 
Rosenau,  104,  130,  182,  428,  550,  630, 

667,  669 

Rosenau  and  Anderson,  103,  104,  132 
Rosenau  and  Brues,  384 


Rosenau,  Lumsden,  and  Kastle,  594 
Rosenau,  Parker,  Francis,  and  Beyer, 

539 

Rosenbach,  300,  302,  307,  308,  318 
Rosenberger,  207 
Rosenow,  449,  453,  456 
Roser,  106 
Ross,  165,  472,  473,  474,  478,  482,  499, 

525,  536 

Ross  and  Milne,  494,  495,  499 
Rossi,  1 60 
Rost,  699,  704 
Rothberger,  607 
Roudoni  and  Sachs,  296 
Rouget  and  Vaillard,  348 
Roux,  24,  98,  128,  170,  215,  218,  222, 

223,  360,  413,  438 
Roux  and  Borrel,  345,  350 
Roux  and  Chamberland,  98,  332 
Roux  and  Martin,  no 
Roux  and  Metschnikoff,  718 
Roux  and  Nocard,  195,  665 
Roux  and  Yersin,  76,  98,  416,  417  419 
Roux,  Pasteur,  and  Chamberland,  363 
Row,  529,  533,  534 
Rowland,  558 

Rowland  and  Macfadyen,  76,  594 
Rudolph,  699 

Ruediger,  311,312,  455,  759 
Ruediger  and  Slyfield,  242 
Ruffer,  578 
Rumpf,  602 
Ruppel,  675 

Russel,  Jordan,  and  Zeit,  593 
Russell  and  Evans,  182 
Russell  and  Hiss,  648 
Russo-Travali  and  Blasi,  420 
Ruzicka,  322 
Ry  mo  witsch  and  Matschinsky ,  40  7 , 409 

SABOURAUD,  752,  753,  754,  756,  757 

Sabrazes,  433 

Saccardo,  759 

Sacharoff,  494 

Sachs  and  Neisser,  139 

Sachs  and  Roudoni,  296 

Saint-Girons,  763 

Salamonsen,  220 

Salant,  86 

Salimbeni  and  Marchoux,  494 

Salkowski,  62,  618 

Salmon,  566,  567,  615,  625,  686 

Salmon  and  Smith,  98,  566,  615,  625, 

626 

Salmonson  and  Madsen,  115 
Sambon,  478,  513 
Sambon  and  Low,  473 
Sanarelli,  536,  615,  627,  628 
Sander,  666 
Sanfelice,  351,  747 
Sattler,  432 
Savage  and  Irons,  607 
Scala,  647 
Schaudinn,  482,   495,  633,  634,  636, 

643,  644 


Bibliographic  Index 


779 


Schaudinn  and  Hoffmann,  24,  69,  718, 

728,  730 
Schenck,  759 
Scherer,  386,  387,  389 
Schereschewsky,  722 
Schering,  150,  179 
Schick,  428,  429 
Schick  and  von  Pirquet,  103 
Schleich,  432 
Schmidt,  386 

Schmitter  and  Nichols,  217,  218 
Schneider,  177,  387 
Schonlein,  755 
Schottelius,  572 
Schottmiiller,  311,  312,  317 
Schroder,  452 

Schroder  and  Van  Dusch,  24,  170 
Schroter,  241 
Schubler,  379 
Schulze,  1 8,  19 
Schumburg  and  Gauss,  31 
Schiitz  andLoffler,  24,  566,  706,  711 
Schiitze  and  Wassermann,  101,  121 
Schwalve,  353 
Schwann  and  Latour,  19 
Sedgwick,  235,  612 
Sedgwick  and  Tucker,  235,  236 
Sedgwick  and  Winslow,  55,  593 
Seifert,  400 
Seiner,  437 
Selter,  200,  714 
Semmelweiss,  22 
Semple  and  Wright,  600 
Sergent  and  Foley,  498 
Shackell,  378 
Shackell  and  Harris,  378 
Shakespeare,  573,  582,  583 
Shakespeare,  Reed,  and  Vaughan,  595 
Shaw,  81 

Shiga,  24,  632,  647,  648,  650,  652 
Shiga  and  Celli,  615 
Shiga  and  Flexner,  651 
Sholly  and  Goodwin,  392 
Shiitz  and  Loffler,  24 
Sicard  and  Widal,  123 
Sievenmann,  44 

Siffer,  McClintock,  and  Boxmeyer,  627 
Silber,  248 

Silverschmidt,  327,  437 
Simon,  315 
Simond,  551 
Simonds,  336 
Simpson,  544,  548 
Sjoo  and  Tornell,  663 
Slyfield  and  Ruediger,  242 
Small,  268 

Small  and  McFarland,  62 
Smirnow,  53 
Smith,  159, 190, 192,  239,  417, 420, 460, 

598,  622,  626,  627,  665,  666 
Smith  and  McCoy,  551 
Smith  and  Salmon,  98,  566,  615,  625, 

626 

Smith  and  Weidman,  328 
Smith  (L.),  161,  197 


Smith  (T.),  59,  60,  103,  124,  127,  200, 
220,  344,  612,  666,  685,  686,  689 

Sobernheim,  575,  578 

Solowiew,  654 

Somers,  609 

Southard  and  Gay,  104 

Sowade,  723 

Soyka,  50 

Spallanzani,  18 

Spengler,  441 

Spiller,  372 

Splendore,  759 

Splendore  and  Lutz,  759 

Spronck,  415,  648,  699 

Starkey,  609 

Steel,  392 

Steele,  71,  602 

Steinhardt  and  Bauzhof,  130 

Steinhardt  and  Besredka,  104 

Steinhardt  and  Poor,  367 

Steinhardt,  Poor,  and  Lambert,  365 

Stein metz  and  Levy,  709 

Stephens,  509 

Stephens  and  Fantham,  506,  509 

Stern,  no,  601,  720,  726 

Sternberg,  24,  60,  106,  235,  250,  252, 
2S3,  304,  325,  343,  444,  459,  53^, 
574,  59°,  592 

Stewart,  149 

Stewart  and  Mitchell,  133 

Stewart,  Graham,   and  Baldwin,  337, 

339 

Sticker,  43,  702 
Stiles,  633 
Stimson,  375,  376 
Stitt,  489,  531 
Stokes,  245 

Stokes  and  Gilchrist,  747,  749 
Stokvis  and  Winogradow,  655 
Stooss,  439 
Strasburger,  71 
Straus,  708,  709 
Strauss  and  Huntoon,  381 
Strehl,  185 
Strickland,  560 
Strong,  558 
Strong  and  Kolle,  558 
Strong  and  Musgrave,  647,  648,  654 
Stuhlern,  457 
Sugai,  701 

Surveyor  and  Boyce,  742,  744 
Swithinbank  and  Neuman,  246 
Swithinbank  and  Newman,  669 
Szekely,  33 
Szemetzchenko,  441 

TAKAKI  and  Wassermann,  09,  345 

Tangl,  417 

Tashiro,  701 

Taube  and  Weber,  691 

Tavel,  41,  316,  351,  592 

Tavel  and  Alvarez,  692 

Taylor  and  Moore,  709 

Tchistowitch,  101,  120,  450 

Tedeschi,  713,  726 


780 


Bibliographic  Index 


Telamon,  444 

Terre  and  Dubarre,  691 

Theiler,  494 

Thelling,  682,  683 

Theodoric,  21 

Thiercelin  and  Lesage,  324 

Thoinot  and  Masselin,  564 

Thomas  and  Cornevin,  96 

Thomas  and  Linton,  509 

Thompson,  644 

Thompson  and  Yates,  507,  509,  612 

Tictin,  498 

Tidswell,  553 

Tiemann  and  Kubel,  608,  609 

Timpe,  190 

Tizzoni  and  Centanni,  683 

Todd,  507 

Todd  and  Button,  494,  495,  499,  507 

Todd  and  Kinghorn,  498 

Todd,  Dutton,  and  Christy,  509 

Tornell  and  Sjoo,  663 

Torrey,  399 

Torry  and  Buxton,  106 

Toussaint,  360 

Toussaint  and  Pasteur,  564 

Trambusti,  56 

Traube  and  Gscheidel,  107,  135 

Trendenburg  and  Frankel,  313 

Treskinskaja,  52 

Triboulet,  324 

Trillat,  180 

Trudeau,  682 

Trudeau  and  Baldwin,  680,  681 

Tsiklinsky,  55 

Tsugitani,  637 

Tucker  and  Sedgwick,  235,  236 

Tunnicliff,  434,  435,  436,  437 

Tyndall,  17,  19,  50 

UCKE,  42O 

Uhlenhuth,  121 

Uhlenhuth  and  Xylander,  664 

Ukke  and  Grigorjeff,  332 

Unna,  68,  155,  368,  403,  663,  696 

Uschinsky,  418,  618 

VAILLARD  and  Dopter,  651 

Vaillard  and  Rouget,  348 

Valagussa,  631 

Vallery-Radot,  565 

Van  der  Pluyn  and  Loag,  394 

Van  de  Velde,  305,  316 

Van  de  Velde  and  Denys,  307 

Van  Dusch  and  Schroder,  24,  170 

Van  Ermengem,  159,  247,  575,  576,  721 

Van  Gehuchten  and  Nelis,  372 

Van  Gieson,  365 

Van  Helmont,  17 

Van  Steenberghe  and  Grysez,  73 

Varro,  20 

Vaughan,"  75,  105,  247,  306,  510,  594 

Vaughan  and  Buxton,  124 

Vaughan  and  Cooley,  619 

Vaughan  and  Novy,  58,  247 

Vaughan,  Cooley,  and  Gelston,  75 

Vaughan  (J.  V.),  75 


Vaughan,  Reed,  and  Shakespeare,  595 

Veasy  and  De  Schweinitz,  408 

Vedder,  642 

Vedder  and  Duval,  648 

Veillon  and  Zuber,  333,  434,  437 

Vergbitski,  553 

Verneuil,  340 

Vesley,  Kimla,  and  Poupe,  666 

Viereck,  633,  635,  643 

Vierordt,  571 

Vignal,  37,  694 

Villemin,  656 

Villiers,  575 

Vincent,  433,  434,  437,  741,  742,  743 

Vincentini,  70 

Vincenzi,  441 

Viola  and  Bonome,  53 

Viquerat,  307,  683 

Virchow,  677,  702 

Virgil,  17 

Vogedes  and  Pfeiffer,  578 

Voges  and  Proskauer,  650 

Voll,  123 

Volpino  and  Bertarelli,  721 

von  Diingern,  100,  102,  116,  134 

von  Fodor,  107 

von  Frisch,  715,  716 

von  Leyden,  603 

von  Lindt,  43 

von  Lingelsheim,  304,  310,  386 

von  Lingelsheim  and  Leuchs,  392 

von  Mayer,  351 

von  Pirquet,  427,  679,  726 

von  Pirquet  and  Schick,  103 

von  Szekely,  33 

Vuillemin,  438 

WADSWORTH,  451,  454 

Wagner,  85 

Wagner  and  Giinther,  721 

Walger,  602 

Walker,  119,  253,  636 

Walker  and  Montgomery,  748 

Walker  and  Rideal,  253 

Walz  and  Baumgarten,  682 

Warren,  191,  427,  473 

Wasdin  and  Geddings,  536 

Washbourn,  453,  456 

Wassermann,  24,  99, 121,  279,  280,  283, 

284,  286,  288,  290,  291,  293,  294, 

297,  323,  396,  397,  398,  726,  728, 

73i 

Wassermann  and  Bruck,  726 
Wassermann  and  Citron.  288 
Wassermann  and  Kolle,  37,  40,  42,  no, 

392,  393,  439,  48o,  482,  483,  495, 

496,  547,  696,  697,  757 
Wassermann  and  Schiitze,  101,  121 
Wassermann  and  Takaki,  99,  345 
Wassermann,  Neisser,  and  Bruck,  279, 

281,  283 
Weaver,  434 
Weber  and  Taube,  691 
Wechsberg  and  Neisser,  136,  137,  138, 

305,  307 


Bibliographic  Index 


Weeks,  406,  407,  408,  432 

Weibel,  588 

Weichselbaum,  241,  386,  387,  389,  391 

392,  444,  452,  552,  599 
Weichselbaum  and  Frankel,  328,  458 
Weidman  and  Smith,  328 
Weigert,  24,  114,  144,  146,  354,  413, 

663,  696,  743,  745 
Weigert  and  Gram,  154 
Weil  and  Kitasato,  219 
Weinzirl,  52 
Welch,  69,  no,  119,  300,  332,  333,  339, 

349,  396,  428,  445,  471,  482 
Welch  and  Abbott,  413 
Welch  and  Flexner,  332,  337,  419 
Welch  and  Nuttall,  332,  334,  337 
Welch  and  Robb,  174 
Wellenhof,  429 
Wenyon,  635 
Wernich,  105 
Wernicke,  24,  550,  588 
Wernicke  and  Kraus,  383 
Wernicke  and  Posadas,  748 
Wertheim,  394,  395,  396 
Wesbrook,  424,  425,  608 
Wesbrook  and  Hankin,  357 
Wesenberg,  327 
Westbrook,  416 
Wheeler,  75 
Whitman,  636 
Wickman,  381 
Widal,  122,  590,  603 
Widal  and  Chantemesse,  599,  601,  602, 

647 

Widal  and  Grunbaum,  603 
Widal  and  Sicard,  123 
Wiener  and  Gruber,  578 
Wiens,  619 
Wigura,  69 
Wilder,  542 

Wilder  and  Ricketts,  542 
Willcomb  and  Winslow,  238 
Williams,  324,  337,  366,  367,  419,  637, 

721 

Williams  and  Calkins,  636  . 
Williams  and  Lowden,  364,  368,  370, 

37i 

Williams  and  Parke,  445 
Wilson,  550 

Wilson  and  McDaniel,  424 
Wilson  and  Yersin,  549 
Winckel,  619 
Windsor  and  Wright,  469 
Winkler,  214 

Winogradow  and  Stokvis,  655 
Winogradsky,  63 
Winslow,  69,  618 
Winslow  and  Prescott,  199 
Winslow  and  Sedgwick,  55,  593 
Winslow  and  Willcomb,  238 
Winter-bottom,  506,  515 
Winterintz  and  Doderlein,  71 
Witte,  190,  191,  200,  610,  612 
Wladimiroff,  496 
Wolbach,  437 


Wolbach  and  Binger,  515,  516 

Wolf,  452 

Wolff,  604,  734 

Wolff  and  Eisner,  680,  726 

Wolffhugel  and  Riedel,  570 

Wolfhugel,  237,  238 

Wollstein,  443,  466,  648 

Wollstein  and  Frankel,  443 

Wood,  148,  457,  729 

Wood  and  Hiippe,  362 

Woodhead,  32,  739 

Woodward,  631 

Wright,  163,  165,  218,  220,  221,  237, 
238,  240,  241,  263,  264,  265,  267, 
268,  271,  273,  274,  275,  277,  307, 
396,  468,  532,  594,  600,  601,  643, 
713,  736,  738,  744,  746 

Wright  (A.  E.),  97,  3°7,  682 

Wright  and  Brown,  733,  734 

Wright  and  Douglas,  107,  270,  277 

Wright  and  Mallory,  163,  165,  220,  386 

Wright  and  Semple,  600 

Wright  and  Windsor,  469 

Wright  (J.  H.),  276,  533,  744,  745 

Wunschheim  and  Fischel,  no 

Wiirtz,  606,  618,  623 

Wyman,  544,  549 

Wynekoop,  466 

Wyse-Lauzun,  763 

Wyssokowitsch  and   Zabolotny,    5151, 
559 

XYLANDER  and  Uhlenhuth,  664 

YAMANOUCHI  and  Levaditi,  280 
Yates  and  Thompson,  507,  509,  612 
Yersin,  543,  544,  545,  546,  547,   549, 

552,  556,^59 
Yersin  and  Kitasato,  24 
Yersin  and  Roux,  76,  98,  416,  417,  419 
Yersin  and  Wilson,  545 
Yorke  and  Kinghorn,  513 
Young,  396,  397,  398 
Yung  and  Pictet,  55 

ZABOLOTNY  and  Wyssokowitsch,  551, 

559 

Zaufal,  452 
Zeit,  53,  712 

Zeit,  Jordan,  and  Russel,  593 
Zenker,  149,  155,  165,  368,  369 
Ziegler,  631 

Ziehl,  151,  157,  590,  660,  661 
Zieler,  155 

Zimmermann,  240,  241 
Zinno,  415 
Zinsser,  218,  460 
Zinsser  and  Hiss,  36, 188,  310,  319,  387, 

396,  447,  448,  456,  589,  614,  708, 

749 

Zinsser,  Hopkins,  and  Gilbert,  724 
Zopf,  33,  241,  457 
Zuber  and  Veillon,  333,  434,  437 
Zupinski,  215 
Zupnik,  346,  347 
Zur  Nedden,  409 


INDEX 


ABBOTT'S  method  of  staining   spores, 

156 

Abderhalden  reaction,  140 
dialysis  test,  140 
optical  test,  140 
technic  of,  142 
Abscess  of  liver  in  amebic  dysentery, 

644 

Acetic  fermentation,  58 
Achorion,  41 

schonleinii,  41,  755 
cultivation,  757 

Krai's  method,  757 
pathogenesis,  758 
Acid,  carbolic,  as  disinfectant,  178 

tuberculinic,  676 
Acids  as  disinfectants,  177 

production  of,  by  bacteria,  60 
Acquired  immunity,  91 

passive,  98 
Actinodiastase,  107 
Actinomyces,  38 
bovis,  732 

cultivation,  734 
distribution,  732 
general  characteristics,  732 
lesions  produced  by,  740 
metabolism,  738 
morphology,  733 
pathogenesis,  738 
temperature,  738 
virulence,  738 
grain,  734 
madurae,  741 
cultivation,  742 
cultural  characteristics,  746 
general  characteristics,  741 
lesions  produced  by,  743 
morphology,  742 
pale  variety,  742 
mode  of  infection  with,  739 
Actinomycosis,  732 
lesions,  740,  743  _ 

mode  of  infection,  739 
Active  immunity,  89 
Acute  anterior  poliomyelitis,  381 
avenues  of  infection,  385 
cause,  381 
characteristics,  382 
histological  changes,  382 
immunization  against,  385 
possible  infective  agent,  384 
transmission,  384,  385 
virus,  382' 
contagious  conjunctivitis,  406 


Adami  and  Chapin's  method  of  isola- 
tion of  Bacillus  typhosus,  608 
Addiment,  101,  118 
Adhesion  cultures,  212 
Aerobes,  51 
Aerobic  bacteria,  51 
Aerogens,  57 

African  lethargy,  506.     See  also  Sleep- 
ing sickness 
Agar-agar,  193 
blood,  195 
culture,  211 
glycerin,  195 
preparation  of,  193 
Agglutination,  122 

test  in  diagnosis  of  sporotrichosis,  764 

technic  of,  124 
Widal  reaction  of,  603 
Agglutinins,  116,  122 
Agglutometer,  603 
Aggressins,  84,  119 
Ague-cake,  486 
Air,  bacteria  in,  50,  234 

quantitative  estimation  by  Hesse's 

method,  234 
by  Petri's  method,  235 
by  Sedgwick's  method,  235 
bacteriology  of,  234 
Air-examination,    Petri's    sand    filter 

for,  236 
Sedgwick    and    Tucker's    expanded 

tube  for,  236 

Alcoholic  fermentation,  57 
Aleppo  boil,  531 
Alexines,  108,  118,  135 
Alkali-albuminate,  Deycke's,  197 
Alkalies  as  disinfectants,  177 

production  of,  by  bacteria,  60 
Allergia,  103 
Altmann's  syringe,  223 
Amboceptor,  dose,  in  Wassermann  re- 
action, 286 

hemolytic,    for    Wassermann    reac- 
tion, 284 
unit,      in      Wassermann      reaction, 

286 
Ameba,  parasitic,  reproduction  cycle 

of,  635 

Amebadiastase,  107 
Amebic  dysentery,  633 

liver  abscess  in,  644 
American  sleeping  sickness,  518 
diagnosis,  522 
prophylaxis,  523 
transmission,  521 


783 


784 


Index 


American   trypanosomiasis,  518.     See 

also  American  sleeping  sickness 
Amoeba  coli,  633 

rhizoppda,  633 
dysenteriae,  633 
Anaerobes,  51 
optional,  51 

Anaerobic  bacteria,  cultivation  of,  215 
by    absorption     of    atmospheric 

oxygen,  215,  217 
by  displaceme  t  of  air  by   inert 

gases,  215 
by    exclusion    of    atmospheric 

oxygen,  215,  219 
by  formation  of  vacuum,  215 
by   reduction  of  oxygen,    215 

218 
cultures,     Botkin's    apparatus    for 

making,  217 
Buchner's  method  of  making,  217, 

219 

Frank  el's  method  of  making,  216 
Hesse's  method  of  making,  219 
Liborius'  tube  for,  216 
Nichols  and  Schmitter's  method 

of  making,  217 
Novy's  jars  for,  216 
Salamonsen's  method  of  making, 

220 

use  of  hen's  eggs  for,  220 
Wright's  method  of  making,  218, 

220 

Zinsser's  method  of  making,  218 
Anaphylactin,  104 
Anaphylaxis,  103 

passive,  105 
Anesthetic  leprosy,  703 
Angina,  Vincent's,  433 
Animal  holder,  guinea-pig,  225 
Latapie's,  225 
mouse,  225 

inoculation  with  malarial  parasites, 
f  486 

Animalculae,  26 

Animals,  experimentation  upon,  222 
method  of  making   injections  into, 

224 

of  securing  blood  from,  226 
post-mortems  on,  228 
typhoid  fever  in,  599 
Anjeszky's  method  of  staining  spores, 

157 

Anopheles  maculipennis,  487,  488 
larva  of,  492 
pupa  of,  492 
Anthracin,  357 
Anthrax,  352 

bacillus  of,  352.     See  also  Bacillus 

anthmcis 

bacteriologic  diagnosis,  361 
lesions,  359 

malignant  pustule  of,  359 
prodigiosus  powder  for,  361,  362 
sanitation  in,  362 
serum  treatment,  361 


Antianaphylactin,  105 

Antibiosis,    influences    on    growth    of 

bacteria,  54 
Antibody,  97 
Antiferments,  116 
Antiformin  as  disinfectant,  176 

for  isolation  of  tubercle  bacillus,  663 
Antigen,  97 

syphilitic,  279 

titration  of,  in  Wassermann  reac- 
tion, 288 

Anti-immune  bodies,  138 
Antiborper,  426 
Antiphthisin,  680 
Antipneumococcus  serum,  456 
Antirabic  serum,  380 
Antisepsis,  23 
Antiseptics,  167 

action  of,  results,  251 

determination  of  value,  251 

inhibition  strengths  of,  180 
Antistreptococcus,  serum,  316 
Antistreptokplysin,  315 
Antitoxin,  diphtheria,  128,  426.     See 
also  Diphtheria  antitoxin 

hypersensitivity  to,  127 

tetanus,  131,  349 
Antitoxins,  125 
Antitubercle  serums,  683 
Antivenene,  100,  132 
Anti-venomous  serum,  132 
Argas  miniatus,  494 
Army-fever,  540 
Arnold's  steam  sterilizer,  171 
Aromatics,  production  of,  by  bacteria, 

62 

Arthrospores,  33 
Ascococcus,  35 
Ascomycetes,  40 
Asiatic  cholera,  568 

discovery  of  specific  organism,  569 
distribution,  568 
history,  568 
prophylaxis,  579 
sanitation,  580 
serum  treatment,  579 
Aspergillus,  42 

flavus,  43 

fumigatus,  43 

malignum,  43 

nidulans,  43 

niger,  44 

subfuscus,  44 
Association,  influences  of,  on  growth 

of  bacteria,  54 

Atrophic  spinal  paralysis,  381 
Auditory  meatus,  external  bacteria  in, 

69 
Autoclave,  modern,  172 

sterilization  in,  171 
Autogenous  vaccines,  265 

BABES  and  Cornil's  method  of  staining 

Spirillum  cholerae  asiaticae,  572 
Babes-Ernst  granules,  31 


Index 


785 


Babes,  tubercles  of,  372 
Bacillary  dysentery,  647 
diagnosis,  651 
lesions,  651 
serum  treatment,  652 
emulsion,  682 
Bacilli,    coli-typhoid,    gram-negative, 

table  of,  650 
in  Ohio  river  water,  classification  of, 

240,  241 

paratyphoid,  614 
resembling  tubercle  bacillus,  692 
typhoid  bacillus,  613 

meat-poisoning  group,  614 
pneumonic      or      psittacosis 

group,  614 

table  for  differentiation,  615 
typhoidal  group,  614 
Bacillus,  35 

aerogenes  capsulatus,  332 
cultivation,  334 
distribution,  333 
general  characteristics,  332 
groups  of,  336 
metabolic  products,  336 
morphology,  333 
pathogenesis,  336 
sources  of  infection,  337 
staining,  334 
vital  resistance,  336 
anthracis,  352 

avenues  of  infection,  357 
bacilli  resembling,  362 
cultivation,  355 
general  characteristics,  352 
isolation,  354 

metabolic  products,  356   i/ 
morphology,  353 
motility,  354 
pathogenesis,  357 
sporulation,  353 
staining,  354 
vaccination,  360 
virulence,  360 
vital  resistance,  356 
avicidum,  564 
avisepticus,  564 

Bordet-Gengou     and     bacillus     in- 
fluenzae,    differences    between, 

443 

cultivation,  442 

isolation,  442 

metabolic  products,  443 

morphology,  441 

pathogenesis,  443 

staining,  442 
botulinus,  59,  247 
butter,  693 
butyricus,  693 
capsulated  canal- water,  457 
capsulatus  mucosus,  457,  458.     See 

also  Pneumococcus 
capsule,  458 
cholerae,  564 

gallinarum,  564 

50 


Bacillus  cholera?  gallinarum,  cultiva- 
tion, 564 

general  characteristics,  564 
immunity,  against,  565 
lesions,  565 

metabolic  products,  564 
morphology,  564 
pathogenesis,  ^64 
staining,  564 
vital  resistance,  564 
coli  communior,  618 
communis,  616 

amount  in  sewage,  623 
cultivation,  617 
distribution,  616 
general  characteristics,  616 
immunization  against,  621 
in  drinking  water,  239,  622 

Wiirtz's  method  of  detect- 
ing, 623 
in  water,  MacConkey's  medium 

for  detecting,  612 
Smith's  method  of  detecting, 

239. 

metabolic  products,  618 

morphology,  616 

pathogenesis,  619 

presumptive  test  for,  242 

staining,  617 

toxic  products,  619 

typhoid  bacillus  and,  differential 
diagnosis,  604, 621, 622 
cultural,  605 
serum,  605 

virulence,  620 

vital  resistance,  618 
comma,  570 
cuniculicida,  564 
diphtherias,  411 

bacilli  resembling,  429 
bacteria  associated  with,  420 
bacteriologic  diagnosis,  425 
chief  types,  424 
contagion  from,  424 
cultivation,  413 

LofBer's  method,  415 
general  characteristics,  411 
habitat,  419,  421 
infection,  intraperitoneal,  418 

intrapleural,  418 
metabolic  products,  417 
morphology,  411 
mucous    membrane    inoculations, 

418 

occurrence  in  healthy  throats,  425 
pathogenesis,  418 
seats  of  infection  by,  421 
specificity,  423 
staining,  413 

Neisser's  method,  413 

with  Loffler's  alkaline  methyl- 

ene  blue,  413 

subcutaneous  inoculation,  418 
toxin,  417 
vital  resistance,  416 


y86 


Index 


Bacillus  Ducreyi,  403 

cultivation,  404 

general  characteristics,  403 

morphology,  403 

pathogenesis,  405 

staining,  404 

vital  resistance,  405 
dysenteriae,  632,  647 

cultivation,  649 

general  characteristics,  649 

Hiss-Russell  variety,  648 

lesions  caused  by,  651 

metabolic  products,  649 

morphology,  649 

pathogenesis,  651 

Shiga-Kruse  variety,  648 

staining,  649 

varieties,  648 

vital  resistance,  651 
enteritidis,  624 

cultivation,  624 

general  characteristics,  624 

lesions,  624 

morphology,  624 

pathogenesis,  624 

sporogenes,  332 

staining,  624 
faecalis  alkaligenes,  624 
fusiformis,  433 

cultivation,  434 

morphology,  436 

pathogenesis,  437 

spirochaeta  vincenti  and,  relation, 

.434 

geniculatus,  70 
Hofmanii,   429.     See  also   Bacillus 

pseudodiphtheria 
icteroides,  cultivation,  628,  629 

distribution,  627 

general  characteristics,  627 

metabolism,  628 

morphology,  627 

pathogenesis,  628 

staining,  627 

vital  resistance,  628 
influenzas,  462 

Bordet-Gengou  bacillus  and,  dif- 
ferences between,  443 

cultivation,  463 

general  characteristics,  462 

immunity  against,  465 

isolation,  463 

morphology,  462 

pathogenesis,  465 

pseudo-,  466 

specificity,  464 

staining,  462 

vital  resistance,  464 
leprae,  6.95 

cultivation,  696 

Clegg's  method,  699 
Duval's  method,  700 
Rost's  method,  699 

etiology,  695 

general  characteristics,  695 


Bacillus   leprae,  lesions   produced   by, 

702 

morphology,  695 
pathogenesis,  701 
staining,  696 
mallei,  706 

cultivation,  709 

distribution,  706 

general  characteristics,  706 

isolation,  708 

lesions  produced  by,  711,  712 

metabolic  products,  710 

mallein,  711 
morphology,  706 
pathogenesis,  711,  717 
staining,  707 

Kiihne's  method,  707 
^Loffler's  method,  707 
virulence,  714 
vital  resistance,  708 
melitensis,    467.     See    also    Micro- 
coccus  melitensis 
Moeller's  grass,  693 
neapolitanus,  616.     See  also  Bacillus 

coli  communis 
cedematis  maligni,  329 
cultivation,  329 
distribution,  329 
general  characteristics,  329 
immunity,  332 
lesions,  331 

metabolic  products,  331 
morphology,  329 
pathogenesis,  331 
staining,  329 
vital  resistance,  330 
of  Asiatic   cholera,   568.     See  also 

Spirillum  cholera  Asiatics 
of  Bordet-Gengou,  441 
of  Biiffelseuche,  566 
of  diphtheria,  411.     See  also  Bacil- 
lus diphtheria 
of  Ducrey,  403.     See  also  Bacillus 

ducreyi 
of     Klebs-Lofner,     411.     See     also 

Bacillus  diphtheria 
of  Koch-Weeks,  406 
association,  408 
cultivation,  407 
general  characteristics,  406 
morphology,  407 
pathogenesis,  408 
staining,  407 
of  Morax-Axenfeld,  408 
cultivation,  408 
morphology,  408 
pathogenesis,  409 
staining,  408 
of  rabbit  septicemia,  564 
of     swine-plague,     566.     See     also 

Bacillus  suisepticus 
of  syphilis,  718.     See  also  Trepan- 

ema  pallidum 

of    typhoid    fever,    589.     See    also 
Bacillus  typhosus 


Index 


787 


Bacillus  of    Weeks,    406.      See  also 

Bacillus  of  Koch-Weeks 
of  Wildseuche,  566 
of  zur  Nedden,  409 
Oppler-Boas,  70 
paracolon,  614 
pestis,  543 

cultivation,  546 

experimental  infection  with,  550 
general  characteristics,  543 
mode  of  infection  with,  551 

by  cutaneous  inoculation,  551 
by  inhalation,  551 
by    intraperitoneal    inocula- 
tion, 554 
by   intravenous    inoculation, 

554 

by      subcutaneous      inocula- 
tion, 551 
metabolism,  550 
morphology,  546 
staining,  546 
thermal  death-point,  550 
virulence,  556 
vital  resistance,  549 
phlegmone  emphysematose,  332 
piscicidus,  248 
proteus  vulgaris,  325 
cultivation,  325 
distribution,  325 
general  characteristics,  325 
metabolic  products,  327 
morphology,  325 
pathogenesis,  327 
staining,  325 

pseudodiphtheria,  chemistry,  431 
cultivation,  430 
differentiation  from  Bacillus  diph- 

theriae,  423,  429 
morphology,  430 
pathogenesis,  431 
staining,  430 
pseudodysentery,  648 
pseudoglanders,  714 
pseudo-influenza,  466 
pseudotetanus,  351 
pseudotuberculosis,  694 
cultivation,  694 
morphology,  694 
pathogenesis,  694 
psittacosis,  625 
cultivation,  625 
differentiation,  625 
general  characteristics,  625 
isolation,  625 
metabolic  products,  625 
morphology,  625 
pathogenesis,  625 
pyocyaneus,  321 
distribution,  321 
general  characteristics,  321 
isolation,  322 
metabolic  products,  323 
morphology,  321 
staining,  322 


Bacillus  rhinoscleromatis,  715 
septicus  sputigenus,  445 
smegmatis,  692 
cultivation,  692 
morphology,  692 
pathogenesis,  692 
staining,  692 
suipestifer,  625 
agglutination,  627 
cultivation,  626 
general  characteristics,  625 
metabolic  products,  626 
morphology,  626 
pathogenesis,  627 
vital  resistance,  626 
suisepticus,  566 
cultivation,  566 
general  characteristics,  566 
lesions  from,  566 
morphology,  566 
staining,  566 
vital  resistance,  566 
tetani,  340 

bacilli  resembling,  351 
cultivation,  342 
distribution,  340 
general  characteristics,  340 
immunity  against,  349 
isolation,  340 
metabolic  products,  344 
staining,  340 
vital  resistance,  343 
tuberculosis,  656 
agglutination,  683 
appearance  of  cultures,  668 
avium,  690 

cultivation,  690 
morphology,  690 
pathogenesis,  691 
staining,  690 
thermic  sensitivity,  691 
bacilli  resembling,  691 
bovis,  685 

lesions  produced  by,  686 
metabolic  products,  685 
morphology,  685 
pathogenesis,  685 
staining,  685 
vegetation,  685 
channels  of  infection  for,  669 

gastro-intestinal  tract,  670 
•  placenta,  669 
respiratory  tract,  669 
sexual  apparatus,  670 
wounds,  671 
chemistry,  675 
cultivation,  665 

Koch's  method,  665 

Nocard    and    Roux's    method, 

665 
Proskauer  and  Beck's  method, 

667 

distribution,  657 
effect  of  tuberculin  on,  677 
general  characteristics,  656 


788 


Index 


Bacillus    tuberculosis   in   sections  of 
tissue,  Ehrlich's  method  of 
staining,  663 
Gram's  method  of  staining, 

663 

Unna's  method  of   staining, 
.  663 

isolation,  antiformin  for,  663 
culture  tube  for,  666 
Dorset's  method,  666 
Frugoni's  method,  667 
Pawlowski's  method,  666 
Smith's  (T.)  method,  666  ] 
lesions  caused  by,  671 
morphology,  657 
pathogenesis,  669 
reaction,  668 
relation  to  oxygen,  669 
staining,  658 

Ehrlich's  method,  66 1 

for  sections,  663 
Gabbett's  method,  66 1 
Gram's  method  for  sections,  663 
in  feces,  663 
in  sputum,  659,  660 
in  urine,662 

Koch-Ehrlich  method,  66 1 
Unna's  method  for  sections,  663 
ZiehPs  method,  66 1 
temperature  sensitivity,  669 
toxic  products,  676 
virulence,  674 
typhi  murium,  628 

destruction  of  mice  by,  630 
general  characteristics,  628 
isolation,  629 
morphology,  629 
pathogenesis,  629 
staining,  629 
typhosus,  589 

bacilli  resembling,  613 

meat-poisoning  group,  614 
pneumonic      or      psittacosis 

group,  614 

table  for  differentiation,  615 
typhoidal  group,  614 
colon    bacillus    and    differential 
diagnosis,  604, 621,622 
cultural,  605 
serum,  605 
cultivation,  591 
.Eisner's  method,  605 
Piorkowski's  method,  608 
Remy's  method,  606 
Rothberger's  method,  607 
Wiirtz  and  Kashida's  method, 

606 

distribution,  589 
effect  of  chemic-  agents  on,  593 

of  cold  on,  593 
general  characteristics,  589 
Hiss'  method  of  cultivation,  607 
i  n  blood,  598 
in  feces,  604 
in  milk,  594 


Bacillus  typhosus  in  oysters,  595 
in  sputum,  598 
in  urine,  598 
in  vegetables,  595 
invisible  growth,  592 
isolation,  591 

Adami  and   Chapin's   method, 

608 

Beckman's  method,  608 
Buxton  and  Coleman's  method, 

6n 

Drigalski-Conradi's  method,  608 
Endo's  method,  610 
Jackson's  method,  612 
Loffier's  method,  611 
MacConkey's  method,  612 
Peabody  and  Pratt's  method, 

611    ' 

Petkowitsch's  method,  608 
Starkey's  method,  609 
metabolic  products,  593 
mode  of  infection  with,  594 
morphology,  590 
pathogenesis,  595 
plating,  Capaldi's  medium  for,  610 

Hesse's  medium,  610 
staining,  590 

Ziehl's  method,  590 
toxic  products,  593 
vital  resistance,  592 
welchii,  332 
xerosis,  431 
chemistry,  432 
cultivation,  432 
morphology,  432 
pathogenesis,  432 
Y,  648 

Bacteremia,  79 
Bacteria,  26 
aerobic,  51 
anaerobic,  51 

cultivation     of,     215.     See     also 

Anaerobic  bacteria,  cultivation  of 

associated  with  diphtheria  bacillus, 

420 

with  suppuration,  299 
Brownian  movement,  32 
capsule  of,  31 
Chester's  synopsis  of  groups  of,  231- 

233 

chromogenic,  61 

colonies  of,  204,  208 

combination  of  nitrogen  by,  64 

counting  of,  by  Willcomb's  method, 

238 

by  Winslow's  method,  238 
by  Wright's  method,  238 
Frost's  plate  counter  for,  239 
Wolf  hug  el's  apparatus  for,  237 

cultivation  of,  187 

determination    of    thermal    death - 
point  of,  249 

discovery  of,  18 

distribution  of,  50 

effect  on  foods  of,  57 


Index 


789 


Bacteria,  fission  of,  32 

flagella  of,  31 

higher,  36^ 

identification  of  species  of,  230 

in  air,  50,  234 

quantitative  estimation  by  Hesse's 

method,  234 
by  Petri's  method,  235 

in  bladder,  71 

in  butter,  246 

in  conjunctiva,  69 

in  dental  caries,  70 

in  external  auditory  meatus,  69 

in  foods,  245 

in  intestine,  70 

in  larynx,  72 

in  lungs,  72 

in  meat,  246 

in  milk,  245 

in  mouth,  69 

in  nose,  71 

in  oysters,  246 

in  sections  of  tissue,  observation  of, 
149 

in  skin  and  adjacent  mucous  mem- 
branes, 68 

in  soil,  243 

Frankel's  method  of  estimation, 

243 

in  stomach,  70 
in  trachea,  72 
in  urethra,  71 
in  uterus,  71 
in  vagina,  71 
in  vegetables,  243 
in  water,  50,  237 
number  of,  238 

method  of  determining,  237 
influences  of  antibiosis  on  growth  of, 

54 

of  association  on  growth  of,  54 
of  chemic  agents  on  growth  of,  56 
of  electricity  on  growth  of,  53 
of  food  on  growth  of,  51 
of  light  on  growth  of,  52 
of  metabolism  on  growth  of,  57 
of  moisture  on  growth  of,  52 
of  movement  on  growth  of,  54 
of  oxygen  on  growth  of,  50 
of  reaction  on  growth  of,  52 
of  symbiosis  on  growth  of,  54 
of  temperature  on  growth  of,  55 
of  x-ray  on  growth  of,  53 

invasion  of  body  by,  78 

invasive  power  of,  75 

isolation  of,  204 

liquefaction  of  gelatin  by,  60 

measurement  of,  166 

morphology  of,  34 

motility  of,  32 

non-chromogenic,  61 

non-pathogenic,  64 

nucleus  of,  31  ' 

number  causing  infection,  81 

pathogenic,  64 


Bacteria,  peptonization  of  milk  by,  64 
photographing,  166 
polar  granules  of,  31 
preparations   for   general   examina- 
tion of,  146 

production  of  acids  by,  60 
of  alkalies  by,  60 
of  aromatics  by,  62 
of  disease  by,  64 
of  enzymes  by,  65 
of  nitrates  by,  63 
of  odors  by,  62 
quantitative  estimation  of,  by  Sedg- 

wick's  method,  235 
reduction  of  nitrates  by,  63 
reproduction  of,  32 
saprophytic,  66 
size  of,  27 
sporulation  of,  32 

staining  of,  146.     See  also  Staining 
structure  of,  30 
study  of  living,  144 
thermophilic,  55 

transplantation    of,    from    culture- 
tube  to  culture-tube,  technic  of,  203 
units  of  measurement,  27 
Bacterial  suspension,  270 

standardization  of,  271 
Bacterination,  95 
Bacteriology,  evolution  of,  17] 
of  air,  234 
of  foods,  245 
of  soil,  243 
of  water,  237 
Bacteriolysis,  135 
technic  of,  136 
Bacteriolytic  serums,  therapeutic  uses 

of,  138 

Bacterio- vaccination,  conditions  neces- 
sary to  success  in,  264 
Bacterio-vaccines,  263 
Bacterium,  35 

coli  dysenteriae,  647 

pneumonias,    445,    457.     See    also 

Pneumococcus 
termo,  325 
Bagdad  boil,  531 
Bain  fixateur,  159 

reducteur  et  reinforgateur,  160 
sensibilisateur,  160 
Balantidium  coli,  652 
cultivation,  654 
habitat,  654 

inoculation  of  animals  with,  654 
lesions  caused  by,  654 
morphology,  652 
motility,  653 
pathogenesis,  654 
staining,  653 
diarrhea,  652 
lesions,  654 
transmission,  655 
Barber's  itch,  754 

Bass  and  Johns'  method  of  cultivating 
malarial  parasites,  484 


790 


Index 


Bazillenemulsion,  682 

Beck  and  Proskauer's  method  of  cul- 
tivation of  tubercle  bacillus,  667 

Beckman's    method    of    isolation    of 
Bacillus  typhosus,  608 

Behring's  method  of  determining  po- 
tency of  diphtheria  serum,  128 

Berk ef eld  filter,  174 

Bichlorid  of  mercury  as  disinfectant, 
176  ^ 

Biologic  contributions,  17 

Biology  of  bacteria,  50 

Biondi-Heidenhain  stain  for  protozoa, 
.165 

Biscra  boil,  531 
button,  531 

Black  death,  543.     See  also  Plague 
molds,  41 

plague,  543.     See  also  Plague 
sickness,  525.     See  also  Kala-azar 

Block  for  subculture  tubes,  257 

Bladder,  bacteria  in,  71 

Blastomyces  dermatitidis,  747 
cultivation,  749 
lesions  caused  by,  751 
pathogenesis,  751 
staining,  749 

Blastomycetes,  38 

Blastomycetic  dermatitis,  747 

Blastomycosis,  747 
specific  organism,  749 
lesions,  751 
transmission,  751 

Blood  agar-agar,  195 

Keidel  tube  for  collecting,  281 
method    of    obtaining   for  Wasser- 

mann  reaction,  282 
from  animals,  226 
phagocytic  power  of,  270 
pipet,  special,  274 
typhoid  bacilli  in,  598 

Blood-corpuscles  for  Wassermann  re- 
action, 283 

Blood-culture  in  typhoid  fever,  603 

Blood-serum,  alkaline,  197 
as  culture- medium,  195,  211 
Koch's    apparatus    for   coagulating 

and  sterilizing  of,  196 
mixture,  LofHer's,  197 
therapy,  24,  222 

Bodies  of  Negri,  363.     See  also  Neuror- 
rhyctes  hydrophobia 

Body,  invasion  of,  by  bacteria,  78 
Leishman-Donovan,  525.     See  also 

Leishmania  donovani 
lice,  503 

Boil,  Aleppo,  531 
Bagdad,  531 
Biscra,  531 
Delhi,  531 
Jericho,  531,  53 2 

Bordet-Gengou  bacillus,  441 
phenomenon,  139 

Botkin's    apparatus    for    making    an- 
aerobic cultures,  217 


Botulism,  247 

Botulismus,  59 

Bouillon  as  culture-medium,  190 
preparation  from  fresh  meat,  190 

from  meat  extract,  191 
sugar,  192 

Bovine  tuberculosis,  685 
tuberculin  test  for,  689 

Broncho-pneumonia,  461 

Broth  nitrate,  63 

Brownian  movement  of  bacteria,  32 

Buboes  in  plague,  544 

Bubonic  plague,  543.     See  also  Plague 

Buchner's  method  of  making  anaerobic 
cultures,  217,  219 

Buerger's  method  of  isolation  of  Dip- 
lococcus  pneumonias,  447 

Buffelseuche,  bacillus  of,  566 

Bulbs,  Sternberg's,  250 

Buret  for  titrating  media,  188 

Burri's  India  ink  method  of  identi- 
fying Treponema  pallidum,  721 

Buton  d'Orient,  531 

Butter  bacillus,  693 
bacteria  in,  246 

Button,  Biscra,  531 

Butyric  acid  fermentation,  58 

Buxton  and  Coleman's  method  of  iso- 
lation of  Bacillus  typhosus,  611 

CALCIUM  carbide  as  disinfectant,  182 
Calmette's  ophthalmo-tuberculin  reac- 
tion, 679 

Canal- water  bacillus,  capsulated,  457 
Canned  goods,  poisoning  from,  248 
Capaldi's  medium  for  plating  bacillus 

typhosus,  610 
Capillary  glass  tubes,  202 
Capsulated  canal- water  bacillus,  457 
Capsule  bacillus,  458 

of  bacteria,  31 
Capsules,  collodion,  228 
Carbolic  acid  as  disinfectant,  178 
Cardinal  conditions  of  infection,  79 
Caries,  dental,  bacteria  in,  70 
Carrasquilla's  leprosy  serum,  704 
Carriers,  typhoid,  595 
Catarrhal  inflammation,  400 

pneumonia,  461 
Celloidin  embedding,  150 
Cells,  lepra,  702 

specific  affinity  of,  for  toxins,  78 
Ceratophyllus  fasciatus,  554,  560 
Cercomonas  intestinalis,  655 
Cerebro-spinal  fever,  386 

meningitis,  386 

bacteriological  diagnosis,  393 
lumbar  puncture  in,  388 
Chamberland  filter.  174 
Chancre,  725 

sporotrichotic,  764 
Chancroid,  403 

specific  organism  of,  403 
Chantemesse's    conjunctival    reaction 

in  typhoid,  604 


Index 


791 


Chapin  and  Adami's  method  of  isola- 
tion of  Bacillus  typhosus,  608 

Charbon,  352  _ 

Cheese-poisoning,  247 

Chemic    agents,    effect    on    Bacillus 
typhosus,  593 

influences  on  growth  of  bacteria,  56 
contributions,  19 

Chester's  synopsis  of  groups  of  bac- 
teria, 231-233 

Chicken-cholera,  564       / 

Chlamydophrys  stercopea,  633 

Chlorin  as  disinfectant,  178 

Cholera,  asiatic,  568.     See  also  Asiatic 

cholera 

changes  in  intestines,  575 
immunity  against   578 
rice-water  discharges  in,  576 
chicken-,  564 
de  poule,  564 
hog-,  625 

Chromogenesis,  61 

Chromogenic  bacteria,  61 

Chromogens,  57 

Cimex  boneti,  521 
lectularius,  521 
rotundatus,  529 

Cladothrix,  37 

Classification  of  protozoa,  44,  45 

Clegg's     method    of    cultivation     of 
Bacillus  leprae,  699 

Clostridium,  33 

Clothing,  disinfection  of,  184 

Coagulins,  116,  120 

Cobralysin,  102 

Cocci,  34 

Coccidioidal  granuloma,  748 

Cold,  effect  of,  on  Bacillus  typhosus 

593 
Coleman    and    Buxton's    method    of 

isolation  of  Bacillus  typhosus,  611 
Coley's  mixture,  316 
Coli-typhoid    bacilli,    Gram-negative, 

table  of,  650 
Collodion  capsules,  228 

sacs,  increase  of  virulence  by  use  of ,  8 1 

preparation  of,  229 
Colon  bacillus,  616.     See  also  Bacillus 

coli  communis 
Colonies  of  bacteria,  204,  208 

counting,     Frost    plate     counter 

for,  239 
in   Esmarch   tubes,    Esmarch's 

apparatus  for,  238 
WolfhiigePs  apparatus  for,  237 
Comma  bacillus,  570 
Complement,  101,  118 
fixation,  139 

test  in  diagnosis  of  glands,  709 

of  sporotrichosis,  764 
for  Wassermann  reaction,  282 
Concentrated  tuberculin,  671 
Conjunctiva,  bacteria  in,  69 
Conjunctiva!  reaction,  Chantemesse's 
in  typhoid,  604 


Conjunctivitis,  acute  contagious,  406 

miscellaneous  organisms  in,  410 
Conorhinus   megistis,    521,    523.     See 

also  Lamus  megistis 
Contagion  from  Bacillus  diphtheriae, 

424 

Coplin's  staining  jar,  151 
Copper  sulphate  as  disinfectant,  177 
Coqueleuch,  441 
Corks,  sterilization  of,  169 
Cover-glass  forceps,  Stewart,  162 
Crab  lice,  504 
Craigia  hominis,  655 

migrans,  655 
Creolin,  179 

Croupous  pneumonia,  444 
Crude  tuberculin,  677 
Cryptobia  borreli,  508 
Ctenocephalus  canis,  561 

felis,  561 

Culex  pipiens,  488 

Cultivation  of  anaerobic  bacteria,  by 
absorption     of     atmospheric 
oxygen,  217 
by     displacement     of     air     by 

inert  gases,  215 
by    exclusion    of    atmospheric 

air,  219 

by  formation  of  vacuum,  215 
by  reduction  of  oxygen,  218 

of  micro-organisms,  187 
Culture  preparations,  museum,  214 
Culture- media,  187 

agar-agar,  193 

alkaline  blood-serum,  197 

blood  agar-agar,  195 

blood-serum,  195 

bouillon,  190 

buret  for  titrating,  188 

Dunham's  solution,  199 

gelatin,  192 

glycerin  agar-agar,  195 

litmus  milk,  199 

Lo  flier's  blood-serum  mixture,  197 

milk,  199 

Parietti's,  609 

Petruschkey's  whey,  199 

potatoes,  197 

potato-juice,  198 

standard  reaction  of,  189 

sterilization  and  protection  of,  170 

sugar  bouillon,  192 
Cultures,  adhesion,  212 

agar-agar,  211 

freshly  isolated,  standardizing,  214 

gelatin  puncture,  209 

in  fluid  media,  212 

inoculation  of,  203 

manipulation  of,  technic,  202 

on  blood  serum,  211 

on  potato,  211 

plate,  20 1,  204 

pure,  201,  208 

special  methods  of  procuring,  212 

shake,  219 


792 


Index 


Cultures,  stab,  209 

study  of,  201 

microscopic,  213 

Cutituberculin  reaction,  Lignieres,  679 
Cytase,  118 
Cytolysis,  134 

technic  of,  135 
Cytoplasm,  46 
Cytotoxins,  133 
Czenzynke's  stain  for  Bacillus  influ- 

enzae,  463 

DEAD,  disinfection  of,  185 
Decrease  of  virulence,  80 
Defensive  ferments,  140 

proteins,  108 

Dejecta,  disinfection  of,  175,  183 
Delhi  boil,  531 
Dental  caries,  bacteria  in,  70 
Denys'  tuberculin,  680 
Dermatitis,  blastomycetic,  747 
Derma tomycosis,  752 
Dermotuberculin   reaction,    von    Pir- 

quet's,  679 
Desmon,  118 

Deycke's  alkali-albuminate,  197 
Diarrhea,  balantidium,  652 
Digestive  apparatus,  infection  through, 

72 

Diphtheria,  411 
antitoxin,  127,  426 

determining  potency  of  serum,  1 28 
dosage,  427 

effect  on  death-rate,  429 
globulin  precipitation  for  concen- 
tration of,  130 

immunization  of  animals,  128 
obtaining  blood  for,  128 
paralysis  from,  427 
preparation  of  serum,  128 
prophylaxis,  427 
treatment  with,  427 
bacillus  of,  411.     See  also  Bacillus 

diphtheria 
bacteriologic  appearance  of  throat, 

420 

characteristics,  419 
course,  420 
lesions,  422 

postmortem  appearance  of  liver/  419 
Schick's  reaction  in,  428 
with  mixed  infection,  422 
Diplobacillen  conjunctivitis,  408 
Diplococcus,  34 

intracellularis  meningitidis,  386 
agglutination,  391 
cultivation,  389 
distribution,  387 
general  characteristics,  386 
identification,  387 
isolation,  388 
metabolic  products,  391 
mode  of  infection  with,  392 
morphology,  387 
pathogenesis,  391 


Diplococcus    intracellularis    meningi- 
tidis, specific  therapy  \\ith,  393 
staining,  388 
vital  resistance,  390 
pneumonias,  444 

animals  susceptible  to,  452 
cultivation,  447 
distribution,  444 
general  characteristics,  444 
identification,  454 
immune  serum  against,  455 
immunity  against,  455 
isolation,  446 

Buerger's  method,  447 
lesions  produced  by,  451 
metabolic  products,  448 
morphology,  445 
pathogenesis,  449 
specificity  of,  452 
staining,  445 

Hiss'  method,  446 
Welch's  method,  445 
toxic  products,  448 
virulence,  453 
vital  resistance,  448 
Disease,  germ  theory  of,  22 
production  of,  64 
tsetse-fly,  512 
Diseases,  infectious,  299 

early  classification  of,  21 
study  of,  20 
Disinfectants,  176 

common,    bactericidal   strength   of, 

181 

determination  of  value  of,  251 
inorganic,  177 
organic,  178 
Dish  forceps,  Petri,  206 
Dishes,  Petri,  206 
Disinfection,  167 

Evans  and  Russell's  method,  182 

Frankforter's  method,  182 

gaseous,  262 

of  air  of  sick-room,  176 

of  clothing,  184 

of  dead,  185 

of  dejecta,  175,  183 

of  furniture,  184 

of  hands,  172,  174 

Lock  wood's  method,  174 
of  instruments,  172 
of  ligatures,  172 
of  patient,  185 
of  room,  182 
of  sick-chambers,  175 
of  sutures,  172 
of  wound,  175 
Robinson's  method,  182 
Distribution  of  bacteria,  50 
Dorset's  method  of  isolation  of  tubercle 

Bacillus,  666 
Dourine,  514 

Drigalski-Conradi's  method  of  isola- 
tion of  bacillus  typhosus,  608 
Drop  infection,  67 


Index 


793 


Ducrey's  bacillus,  403.     See  also  Bacil- 
lus ducreyi 
Dumdum  fever,  525.     See  also  Kala- 

azar 

Dunham's  peptone  solution,  199 
Duval's    method    of    cultivation    of 

Bacillus  leprae,  700 
Dyscrasia,  84 
Dysentery,  631 

amebic,  633 

bacillary,  647 

distribution,  631 

history,  631 

EDEMA,  gaseous,  332 

malignant,  329 

Ehrlich's  lateral-chain  theory  of  im- 
munity, no 

his  explanation  of,  112-117 
method  of  determining  potency  of 

diphtheria  serum,  129 
of  staining  tubercle  bacillus,  66 1 
Electricity,  influences  of,  on  growth  of 

bacteria,  53 
Electrozone,  178 
Elephantiasis  graecorum,  702 
Eisner's    method    of     cultivation    of 

Bacillus  typhosus,  605 
Embedding,  150 
celloidin,  150 
glycerin-gelatin,  151 
paraffin,  150 
Emulsion,  bacillary,  682 
Encystment  of  protozoa,  49 
Endogenous  infections,  68 
Endo's  method  of  isolation  of  Bacillus 

typhosus,  6 10 
Endospores,  32 
Endotoxins,  264 
Entamceba  coli,  634 

table  of  differential  features,  643 
histolytica,  633,  634 
morphology,  634 
relationship  to  Entamceba  tetra- 

gena,  636 
reproduction,  634 
staining,  634 
table     of     differential     features, 

643 

Mallory's  differential  stain  for,  647 
tetragena,  633,  635 
cultivation,  636 
isolation,  636 
lesions,  644 

metabolic  products,  642 
pathogenesis,  644 
relationship  to  Entamceba  tetra- 
gena, 636 

table  of  differential  features.  643 
vital  resistance,  637 
Enteric  fever,  589.     See  also  Typhoid 

fever 

Enzymes,  production  of,  65 
Eosin      and      methylene-blue      stain, 
Mallory's,  155 


Epidemic    cerebro-spinal    meningitis, 
386 

Epitheliolysins,  102 

Erythrasma,  752 

Esmarch's    instrument    for    counting 
colonies  of  bacteria  in  Esmarch 
tubes,  238 
tubes,  207 

Eurotium,  42 

Evans  and  Russell's  method  of  disin- 
fection, 182 

Exhaustion  theory  of  immunity,  105 

Exogenous  infections,  67 

Experimentation  upon  animals,  222 

Extracellular  toxins,  76 

FARCIN  du  bceuf,  37 
Farcy,  711 
Farcy-buds,  711 
Favus,  755 

sculutum  formation,  755 

specific  organism,  755 
Febrile    tropical    splenomegaly,    525. 

See  also  Kala-azar 

Feces,    isolation   of    typhoid    bacillus 
from,  604 

staining  tubercle  bacillus  in,  663 
Ferment,  inflammatory,  23 

putrefactive,  23 
Fermentation,  19,  57 

alcoholic,  57 

acetic,  58 

butyric  acid,  58 

lactic  acid,  58 

Fermentation-tube,  Smith's,  59 
Ferments,  defensive,  140 
Fever,  army-,  540 

enteric,     589.     See     also     Typhoid 
fever 

jail-,  540 

malta,  467 

Mediterranean,  467 

non-malarial    remittent,    525.     See 
also  Kala-azar 

relapsing,  494 

ship-,  540 

splenic,  352 

spotted,  386 

typhoid,  589 

yellow,  536 
Filter,  Berkefeld,  174 

Chamberland,  174 

Kitasato,  174 

Reichel,  174 
Filterable  viruses,  28 
Filtration,  sterilization  by,  172 
Finkler  and  Prior  spirillum,  580 
Fiocca's  method  of  staining  spores,  157 
Fish  tuberculosis,  691 
Fishing,  209 
Fish-poisoning,  248 
Fission  of  bacteria,  32 
Fixateur,  118 
Fixation  of  complement,  139 

test  of  Wassermann  reaction,  291 


794 


Index 


Flagella  of  bacteria,  3 1 

Flagellation,  472 

Flagellates,   harmless,    of   human   in- 
testines, 655 

Fleas,  plague,  559 

transmission  of  plague  by,  554 

Fleischvergiftung,  59 

Flexner  variety  of  dysentery  bacillus, 
648 

Fluid  media,  cultures  in,  212 

Fly,  tsetse,  517 

Fomites,  foods  as,  245 

Food,  influences  on  growth  of  bacteria, 

5i 

poisons,  247 
Foods  as  fomites,  245 
bacteria  in,  245 
bacteriology  of,  245 
effect  of  bacteria  on,  57 
Forceps,  sterilization  of,  169 
Formaldehyd,  180 
Formalin,  179,  181 
Fowl  tuberculosis,  690 
Frambesia  tropica,  729 
diagnosis,  731 
distribution,  729 
history,  729 
specific  organism,  730 
Frankel's    instrument    for    obtaining 

earth  for  bacteriologic  study,  244 
method   of   estimating   bacteria   in 

soil,  243 

of  making  anaerobic  cultures,  216 
Frankforter's  method  of  disinfection, 

182 
Friedlander,    pneumococcus    of,    457. 

See  also  Pneumococcus 
Frost's    plate    counter    for    counting 

colonies  of  bacteria,  239 
Frothy^organs,  338 

Frugoni's   method  of   isolation  of  tu- 
bercle bacillus,  667 
Fungi,  imperfect,  40 
Fungus,  ray-,  732 
Furniture,  disinfection  of,  184 

GABBETT'S  method  of  staining  tubercle 

bacillus,  66 1 
Galactotoxism,  247 
Gamaleia,  spirillum  of,  584,  586 
Gangrene,  hospital,  329 
Gaseous  disinfection,  262 

edema,  332 
Gases,  production  of,  59 

Smith's    method    for     determining 

nature  of,  59 
Gastro-intestinal  tract,  infection  with 

tubercle  bacillus  through,  670 
Gelatin  as  culture-medium,  192 

liquefaction  of,  by  bacteria,  59 

puncture  culture,  209 
Generation,  spontaneous,  doctrine  of, 

17 
Genital  apparatus,  infection  through, 

74 


Germ  theory  of  disease,  22 
Germicides,  167 

determination  of  value  of,  251,  252 
apparatus  for,  253 
culture  media  for,  256 
determining   phenol   coefficient 

in,  258-261 

dilution  of  phenol  and  test  solu- 
tions for,  256 
inoculating  loops  for,  254 
Koch's  method,  253 
modern  method  of  testing,  253 
racks  for  holding  tubes  in,  256 
reagents  for,  253 
solution  to  be  tested,  254 
Sternberg's  method,  253 
test  organism  for,  254 
tubes  for,  256 
water-bath  for,  254 
Germination  of  spores,  34 
Ghoreyeb's   method  of   staining  Tre- 

ponema  pallidum,  719 
Giant-cells  in  tubercles,  672 
Glanders,  706 
bacillus,     706.     See    also    Bacillus 

mallei 
diagnosis,  708 

complement-fixation  test,  709 
McFadyen's  method,  709 
Straus'  method,  708 
immunity  against,  714 
in  human  beings,  713 
lesions,  711,  712 
specific  organism,  706 
Glass  tubes,  capillary,  202 
Glassware,  protection  of,  167 

sterilization  of,  167,  169 
Globulin  precipitation  for  contentra- 

tion  of  diphtheria  antitoxin,  130 
Glossina  bocagei,  518 
fusca,  518 
longipalpis,  518 
longipennis,  518 
morsitans,  513,  518 
pallicera,  518 
pallidipes,  518 
palpalis,  512,  513,  518 
tachinoides,  518 
Glycerin  agar-agar,  195 
Glycerin-gelatin,  embedding,  151 
Golden  staphylococcus,  302 
Goldhorn's    method    of  staining  Tre- 

ponema  pallidum,  719 
Gonococcus,  394 
Gonorrhea,  394 
Gonotoxin,  397 
Gordon's    method    for    detection    of 

Spirillum  choler^e  Asiaticae,  577 
Gram's  method  of  staining  bacteria  in 

tissue,  152,  153 
solution,  152 
Gram-Weigert  stain^  154 
Granuloma  coccidioidal,  748 
Grass  bacillus,  Moeller's,  693 
Guinea-pig  holder,  225 


Index 


795 


Guinea-pig    serum,   titration    of,   for 
Wassermann  reaction,  283 

HAFFKINE  prophylactic  in  plague,  557 
Halogens  as  disinfectants,  178 
Hands,  disinfection  of,  172,  174 

Lock  wood's  method,  174 
Hanging  block,  Hill's,  145 

drop,  145 
Hardening,  149 
Harris'     method    of    staining    Negri 

bodies,  368 
Harris    and    Shackell's    treatment   of 

hydrophobia,  378 
Head  lice,  503 
Heidenhain's  iron   hematoxylin  stain 

for  protozoa,  165 
Heiman's    method    of    cultivation    of 

Micrococcus  gonorrhceae,  396 
Helcosoma  tropicum,  532,  533 
Hemolysis,  101,  133 

technic  of,  134 

Hemolytic    amboceptor,  for    Wasser- 
mann reaction,  284 
serum    for    Wassermann    reaction, 

titration  of,  285 
system,  286 

test  of  Wassermann  reaction,  291 
unit,  286 
Hemorrhagin,  132 

Hen's  eggs,  use  of,  for  anaerobic  cul- 
tures, 220 

Herpes  circinatus,  752 
desquamans,  752 
tonsurans,  752 
Hesse's   medium  for  plating  Bacillus 

typhosus,  6 10 

method    of    making   anaerobic   cul- 
tures, 219 
of     quantitative     estimation     of 

bacteria  in  air,  234 
Higher  bacteria,  36 
Hill's  hanging  block,  145 
Hiss'  inulin-serum-water  test  for  de- 
termining pneumococcus,  455 
method   of    cultivation  of   Bacillus 

typhosus,  607 

of    staining    Diplococcus    pneu- 
monias, 446 
Hiss-Russell  variety  of,  Bacillus  dys- 

enteriae,  648 

Histologic  lesions  in  typhoid  fever,  597 
Histoplasma  capsulatum,  534,  535 
Histoplasmosis,  534 
Historical  introduction,  17 
Hofmann,  bacillus  of,  429.     See  also 

Bacillus,  pseudo-diphtheria 
Hog-cholera,  625 
Hogyes'    treatment    of    hydrophobia, 

374,  378 

Hospital  gangrene,  329 
Host,  66 

susceptibility  of,  84.     See  also  Sus- 
ceptibility 
Hot-air  sterilizer,  169 


Huhnercholera,  564 

Human  malarial  parasites,  478 

human  inoculation  with,  486 
Hydrophobia,  363 
diagnosis,  370 
dumb,  372 

examination  for  Negri  bodies,  371 
histologic  changes  in  nervous  sys- 
tem, 372 

immunization  against,  374 
inoculation  of  rabbits,  371 
pathology,  369 
prophylaxis,  373 
specific  organism,  363 
street  virus,  372 
treatment,  374 

Harris  and  Shackell's  inspissation 

method,  378 
Hogyes  attenuation  method,  374 

dilution  method,  378 
intensive,  scheme  for,  378 
mild,  scheme  for,  377 
Pasteur's  method,  96,  374,  377 
specific,  380 

tubercles  of  Babes  in,  372 
virulence,  372 
Hypnococcus,  508 

ICE-CREAM  poisoning,  247 

Ichthyotoxism,  248 

Identification  of  species  of  bacteria, 

230 

Immune  bodies,  101,  118 
Immunity,  88 

active,  89 
acquired,  91 

acquired,  91 

through  infection,  91 
accidental,  91 
experimental,  92 
through  intoxication,  97 

against  tetanus,  349 

Ehrlich's  lateral-chain  theory  of ,  no 

exhaustion  theory  of,  105 

experimental  investigation  of  prob- 
lems of,  100 

explanation  of,  105 

natural,  89 

passive,  89 
acquired,  98 

relative,  89 

retention  theory  of,  105 

special  phenomena  of,  120 
Imperfect  fungi,  40 
Increase  of  virulence,  80 
Incubating  oven,  213 
Incubator  for  opsonic  work,  276 
Index,  opsonic,  270 
Indol,  production  of,  62 

Salkowski's  test  for,  62 
Infantile  kala-azar,  530 

palsy,  381 
Infection,  avenues  of,  72,  82 

cardinal  conditions  of,  79 

definition  of,  66 


Index 


Infection,  drop,  67 

immunity  acquired  through,  91 

number  of  bacteria  causing,  81 

sources  of,  67 

special  phenomena  of,  120 

through  digestive  apparatus,  72 

through  genital  apparatus,  74 

through  placenta,  74 

through  respiratory  apparatus,  74 

through  skin,  72 
Infections,  endogenous,  68 

exogenous,  67 

mixed,  86 

terminal,  312 
Infectious  diseases,  299 

study  of,  20 

Inflammation,  catarrhal,  400 
Inflammatory  ferment,  23 
Influenza,  462 

bacillus  of,  462.     See  also  Bacillus 
influenza 

diagnosis,  466 
Infusoria,  46 
Infusorial  life,  19 
Injections,  animal,  methods  of  making, 

224 
Inoculating  loops,  254 

device  for  flaming  of,  257 
Inoculation,  92 

early,  for  smallpox,  92 

of  cultures,  203 
Inorganic  disinfectants,  177 
Instruments,  disinfection  of,  172 

sterilization  and  protection  of,  167 
Intermediate  body,  101 
Intermittent  sterilization,  170 
Intestines,  bacteria  in,  70 

human,  harmless  flagellates  of,  655 
Intoxication,  immunity  acquired  bv, 

97 

Intracellular  toxins,  75 
Invasive  power  of  bacteria,  75 
Invisible  viruses,  28 
lodin  terchlorid  as  disinfectant,  178 
Isolation  of  bacteria,  204 
Itch,  barbers',  754 

JACKSON'S    method    of    isolation     of 

Bacillus  typhosus,  612 
Jactationstetanus,  347 
Jail-fever,  540 

avelle  water,  664 

"aw,  lumpy,  732 
"ennerian  vaccination,  93 

ericho  boil,  531,  532 

KALA-AZAR,  525 
diagnosis,  530 
infantile,  530 
lesions, "528 
transmission,  529 
treatment,  530 

Kashida  and  Wiirtz's  method  of  cul- 
tivation of  Bacillus  typhosus,  606 

Keidel  tube  for  collecting  blood,  281 


Keuchhusten,  441 
Kitasato  filter,  174 
Klatschpraparat,  212 
Klebs-Loffler,    bacillus    of,    411.     See 

also  Bacillus  diphtheria 
Knives,  sterilization  of,  169 
Koch-Ehrlich  method  of  staining  tu- 
bercle bacillus,  66 1 
Koch-Weeks  bacillus,  406.     See  also 

Bacillus  of  Koch-Weeks 
Koch's     apparatus     for     coagulating 
blood-serum,  196 

law,  22 

method  of   determining    germicida] 

value,  253 

of  cultivation  of  tubercle  bacillus, 
665 

plate  cultures,  201 

syringe,  222 

tuberculin,  676 
Krai's     method     of     cultivation     of 

Achorion  schonleinii,  757 
Kreotoxism,  247 
Kiihne's  method  of  staining    Bacillus 

mallei,  707 

LABYRINTH,  Starkey's,  Somers'  modi- 
fication, 609 
Lacmoid,  199 

Lactic  acid  fermentation,  58 
La    fievre    typhique,    589.     See    also 

Typhoid  fever 
Laitinen's    method   of   cultivation   of 

Micrococcus  gonorrhoeas,  396 
Lamblia  intestinalis,  655 
Lamus  megistis,  521,  523 
appearance,  523 
breeding  habits,  524 
habitat,  523 
habits,  523 

Larynx,  bacteria  in,  72 
Latapie's  animal  holder,  225 
Latent  tuberculosis,  674 
Lateral-chain    theory    of     immunity, 

Ehrlich's,  no 
Leishman-Donovan    body,    525.     See 

also  Leishmania  donovani 
Leishmania  donovani,  525 
cultivation,  527 
distribution,  528 
evolution,  526 
morphology,  525 
furunculosa,  531 
cultivation,  533 
pathogenesis,  533 
infantum,  529,  530 
Lepra  anaesthetica,  702 
cells,  702 
nodosa,  702 
Leprolin,  704 
Leprosy,  695 
anesthetic,  703 
Carrasquilla's  serum  for,  704 
distribution,  695       •   . 
history,  695 


Index 


797 


Leprosy,  lesions,  702 

sanitation,  704 

specific  therapy,  704 
Leptopsylla  musculi,  560 
Leptothrix,  36 
Leuconostoc,  35 
Leukocidin,  305 
Leukocytes,  washed,  in  opsonic  value 

of  blood,  272 
Levaditi's  method  of   staining  Trepo- 

nema  pallidum,  721 
Leveling  apparatus  for  making  plate 

cultures,  205 
Liborius'  tube  for  anaerobic  cultures, 

216 
Lice,  505 

body,  503 

crab,  504 

head,  503 

r6le  in  transmission  of  typhus  fever, 

542 

transmission  of  plague  by,  553 
Ligatures,  disinfection  of,  172 

sterilization  of,  175 
Light,  influences  on  growth  of  bacteria, 

52 

Lignieres  cutituberculin  reaction,  679 
Liquefaction  of  gelatin  by,  60 
Listerism,  23 

Litmus,  preparation  of,  199 
Litmus-lactose-agar-agar,  606 
Litmus- milk,  199 
Lobar  pneumonia,  444 
Lock-jaw,  340,  347 
Lockwood's  method  of  disinfection  of 

hands,  174 

LofHer's  alkaline  methylene-blue,  151 
blood-serum  mixture,  197 
method  for  detection  of  Spirillum 

cholerae  Asiaticse,  577 
of  isolation  of  Bacillus  typhosus, 

611 

of  staining  Bacillus  mallei,  707 
bacteria,  151 
flagella,  158 
Loops,  inoculating,  254 
Luetin,  724 

reaction,  Noguchi's,  in  diagnosis  of 

syphilis,  727 

Lumbar  puncture,  technic  in,  388,  389 
Lumpy  jaw,  732 
Lungs,  bacteria  in,  72 
Luzzani's   method   of   staining   Negri 

bodies,  369 
Lysin,  101 
Lysol,  179 
Lyssa,  363.     See  also  Hydrophobia 

MACCONKEY'S  method  of  isolation  of 

Bacillus  typhosus,  612 
Macrocytase,  107 
Macrophages,  106 
Madura-foot,  741 
Makrogametes,  477 
Makrogametocyte,  477 


Maladie  du  coit,  514 

Maladie  du  sommeil,   506.     See  also 

Sleeping  sickness 
Malaria,  471 
fever  in,  471 

geographic  distribution,  471 
history  of,  471 
parasites  of,  472 

animal  inoculation,  486 
cultivation,  484 

Bass  and  Johns'  method,  484 
human,  478 

inoculation  with,  486 
paroxysms  of,  471 

pathogenesis,  486 
prophylaxis,  486 
human  beings,  486 
mosquitoes,  486 

relation  of  mosquitoes  to,  473,  488 
Malignant  edema,  329 

poly  adenitis,  543.     See  also  Plague 
pustule,  359 
Mallein,  711 
Mallory's    differential    stain    for  'en- 

tamceba,  647 
method  of  staining,  155 
Malta  fever,  467 

bacteriologic  diagnosis,  468 
treatment,  469 
Manouelian's     method     of     staining 

Treponema  pallidum,  722 
Marino's  stain  for  protozoa,  164 
Mastigophora,  45 
McFadyen's  method  of  diagnosis  of 

glanders,  709 
Meat,  bacteria  in,  246 

extract,     preparation     of     bouillon 

from,  191 
fresh,  preparation  of  bouillon  from, 

190 

Meat-infusion,  190 
Meat-poisoning,  59,  247 
Medical  contributions,  20 
Mediterranean  fever,  467 
Megastomum  intestinalis,  655 
Melanoid  mycetoma,  741,  744 
Meningitis,  cerebrospinal,  386 
Meningococcus,  386 
Mercuric  chlorid  as  disinfectant,  177 
Merismopedia,  34 
Merozoits,  477 
Metabolism,  influences  on   growth  of 

bacteria,  57 
Metazoa,  44 
Metschnikoff's  theory  of  phagocytosis, 

106 

Meyer's  syringe,  222 
Mice,  destruction  of,  by  Bacillus  typhi 

murium,  630 
Micrococci,  34 

Micrococcus  catarrhalis,  388,  400 
cultivation,  401 
general  characteristics,  400 
morphology,  401 
pathogenesis,  401 


Index 


Micrococcus  catarrhalis,  staining,  401 
gonorrhoeas,  394 
cultivation,  395 

Heiman's  method,  396 
Laitinen's  method,  396 
Wassermann's  method,  396 
Wertheim's  method,  395 
Young's  method,  396 
diagnosis     of     gonorrhea     from, 

397  . 

distribution,  394 
general  characteristics,  394 
immunization  against,  399 
isolation,  395 
morphology,  394 
pathogenesis,  398 
staining,  395 
toxic  products,  397 
vital  resistance,  39*6 
melitensis,  467 
cultivation,  467 
general  characteristics,  467 
morphology,  467 
pathogenesis,  469 
staining,  467 
thermal  death  point,  467 
tetragenus,  318 
cultivation,  319 
general  characteristics,  318 
isolation,  319 
morphology,  319 
palhogenesis,  320 
staining,  319 
Microcytase,  107 
Microgametes,  477 
Microgametocyte,  477 
Micro  millimeter,  26 
Micron,  27 

Micro-organisms,  classification  of,  26 
cultivation  of,  187 
measurement  of,  166 
methods  of  observing,  144 
photographing,  166 
specific,  299 
structure  of,  26 
Microphages,  106 
Microscopic  study  of  cultures,  213 
Microspira,  35 
Microsporon,  41 
Miliary  tubercle,  673 
Milk  as  culture-medium,  198 
Bacillus  typhosus  in,  594 
bacteria  in,  245 
peptonization  of,  64 
Milk-poisoning,  247 
Milzbrand,  352 
Mixed  infections,  86 

pneumonias,  461 
Mixture, .  Coley's,  .316 
Moeller's  grass  bacillus,  693 
Moisture,  influences  on  growth  of  bac- 
teria, 52 
Molds,  40 

black,  41 
M  oiler's  method  of  staining  spores,  157 


Morax-Axenfeld    bacillus,     408.     See 

also  Bacillus  of  Morax-Axenfeld 
Morphology  of  bacteria,  34 
Morve,  706 
Mosquitoes,  breeding  habits,  490 

classification,  489 

destruction    of,    in    prevention     of 
malaria,  487 

development  of  larva;,  490 
of  pupae,  490 

habits  of  pupae,  491 

longevity  of  female,  491 

method  of  dissection,  493 

of  infecting  with  malarial  para- 
sites, 493 
of  mounting,  492,  493 

relation  to  malaria,  473,  488 

yellow  fever  and,  537 
Motility  of  bacteria,  32 
Mouse  holder,  225 
Mouth,  bacteria  in,  69 
Movement,   influences    on  growth  of 
bacteria,  54 

of  protozoa,  48 
Mucor,  41 

conoides,  42 

corymbifer,  42 

pusillus,  42 

ramosus,  42 

rhizopodiformis,  42 

septatus,  42 

Mucous  membranes,  bacteria  in,  68 
Muguet,  438 
Muir  and  Ritchie's  method  of  staining 

spores,  156 

Museum  culture  preparations,  214 
Mussel-poisoning,  248 
Mycetoma,  741 

characteristics,  741 

distribution,  741 

melanoma,  741 

melanoid  form,  744 

ochroid,  741 
Mycophylaxins,  108 
Mycosozins,  108 
Mytilotoxism,  248 
Myzorrhynchus  pseudopictus,  488 

NAGANA,  512 

Natural  immunity,  89 

Needles,    platinum,    for    transferring 

bacteria,  202 
Negri  bodies,  363.     See  also  Neuror- 

rhyctes  hydrophobia 
Neisser-Wechsberg  phenomenon,  136, 

137 

Nephrolysins,  102 
Nephrotoxins,  102 
Nessler's  solution,  64 
Neurorrhyctes  hydrophobias,  363 
cultivation,  366 
morphology,  364 
staining,  367 

Harris'  method,  368 
Luzzani's  method,  369 


Index 


799 


Neurorrhyctes  hydrophobias,  staining, 
Reichel  and  Engle's  method, 
369 
Williams        and          Lowden's 

method,  368 
Newman's  method  of  staining  flagella, 

Smith's  modification,  161 
Nichols   and    Schmitter's   method   of 

making  anaerobic  cultures,  217 
Nicolle,  N.  N.  N.  medium  of,  527 

modification  of  Gram's  method,  154 
Nitrate  broth,  63 
Nitrates,  formation  of,  62 

reduction  of,  63 
Nitrobacter,  63 
Nitrogen,  combination  of,  64 
Nitrosococcus,  63 
Nitrosomonas,  63 
N.  N.  N.  medium  of  Nicolle,  527 
Nocard,  bacillus  of,  625 
Nocard  and  Roux's  method  of  culti- 
vating Bacillus  tuberculosis,  665 
Noguchi's  luetin  reaction  in  diagnosis 
of  syphilis,  727 

method  of  cultivation  of  Treponema 
pallid um,  722 

modification  of  Wassermann  reac- 
tion, 294 

Non-chromogenic  bacteria,  61 
Non-malarial    remittent    fever,    525. 

See  also  Kala-azar 
Non-pathogenic  bacteria,  64 
Nose,  bacteria  in,  71 
Novy's  jars  for  anaerobic  cultures,  216 
Nucleus  of  bacteria,  31 

of  protozoa,  48 

OcHROiD|mycetoma,  741 
Odors,  production  of,  by  bacteria,  62 
Oidia,  39 

Oidium  albicans,  39,  438 
cultivation,  439 
fermentation,  440 
immunity  against,  440 
metabolic  products,  440 
morphology,  438 
pathogenesis,  440 
Onychomycosis,  752 
Oocysts,  49,  477 
Ookinetes,  49,  477 
Ophidiomonas,  36 
Ophthal mo-tuberculin    reaction,    Cal- 

mette's,  679 
Opilacao,  519 
Opsonic  index,  270 

determination  of,  277 
negative  phase  of,  277 
positive  phase  of,  277 
theory,  270 
value    of    blood,    requirements    for 

test  of,  270 
serum  in  testing,  273 
washed  leukocytes  in,  272 
work,  incubator  for,  276 
Opsonins,  107,  270 


Opspnizing  pipet,  274 
Optional  anaerobes,  51 
Organic  disinfectants,  178 
Oriental  sore,  531,  533 
Ornithodorus  moubata,  499,  502,  521 

habitat,  504 
savignyi,  499,  501 

habitat,  502 
Oven,  incubating,  213 
Oxygen,  influences  on  growth  of  bac- 
teria, 50 

relation  to  tubercle  bacillus,  669 
Oysters,  Bacillus  typhosus  in,  595 
bacteria  in,  246 

PALUDISM,  471 
Paracolon  bacillus,  614 
Paraffin  embedding,  150 
Parasite,  66 

stomatitis,  438 
Parasites,  malarial,  472 

animal  inoculation  with,  486 
cultivation,  484 
human,  478 

inoculation  with,  486 
pathogenesis,  486 
Parasitic    ameba,    reproductive   cycle 

of,  635 
bacteria,  66 

Paratyphoid  bacilli,  614 
Pariette's  culture  fluid,  609 
Paroxysms  of  malaria,  471 
Passive  anaphylaxis,  105 
immunity,  89 
acquired,  98 

Pasteur-Chamberland  filter,  173 
Pasteurization,  171 
Pasteur  treatment  of  rabies,  96,  378 

schemata  for,  377 
vaccination,  95 
Pathogenesis,  75 
Pathogenic  bacteria,  64 
Pathogens,  57 
Patient,  disinfection  of,  185 
Pawlowski's   method   of   isolation    of 

tubercle  bacillus,  666 
Peabody  and  Pratt's  method  of  isola- 
tion of  Bacillus  typhosus,  611 
Pediculus  capitis,  503,  505 
pubis,  504 
vestimenti,  503,  505 
Penicillium,  44 

Peptone  solution,  Dunham's,  199 
Peptonization  of  milk,  64 
Peroxid  of  hydrogen,  179 
Pertussis,  441 
Pest,  543.     See  also  Plague 
Petkowitsch's  method  for  isolation  of 

Bacillus  typhosus,  608 
Petri  dish,  206 

advantages  of,  207 
forceps,  206 
method  of  quantitative  estimation  of 

bacillus  in  air,  235 
sand  filter,  for  air-examination,  236 


8oo 


Index 


Petruschkey's  whey,  199 

Pfeiffer's  method  of  staining  bacteria, 

u151 
phenomenon,  101 

Phagocytes,  106 

Phagocytic  power  of  blood,  270 

Phagocytosis,    Metschnikoff's    theory 

of,  106 

Phagolysis,  107 

Phenol   coefficient,    technic   of   deter- 
mining, 258-261 
Phenomenon,  Bordet-Gengou,  139 

Neisser-Wechsberg's,  136 

Pfeiffer's,  101 

Theobald  Smith's,  103 
Phlogosin,  305 
Phosphorescence,    production    of,   by 

bacteria,  62 
Photogens,  57 

Phthirius  inguinalis,  504,  505 
Phycomycetes,  40 
Phylaxins,  108 
Pied  de  Madurae,  741 
Pig  typhoid,  625 
Pink  eye,  406 
Piorkowski's  method  of  cultivation  of 

Bacillus  typhosus,  608 
Pipet,  opsonizing,  274 

special  blood,  274 
Pitfield's  method  of  staining  flagella, 

159 

modification,  Smith's,  159 
Pityriasis  versicolor,  752 
Placenta,  infection  through,  74 
with  tubercle  bacillus,  669 
Plague,  543 
buboes  in,  544 
characteristics,  544 
death-rate,  544 
diagnosis,"  555 
experimental  infection,  550 
fleas,  559 

breeding  habits,  560 
life  history,  559 
longevity,  560 

method  of  extermination,  560 
table  for  identification,  563 
varieties,  560,  561,  562 
group  of  micro-organisms,  563 
history,  543 
immunity  against,  557 
active,  557 

Haffkine  prophylactic,  for,  557 
passive,  558 
pneumonia,  461 

postmortem  appearance  in,  555 
prophylaxis,  557 
rat  extermination  in,  557 
sanitation  in;  556 
serum  treatment,  559 
specific  organism,  545 
spread  of,  543 

transmission  by  fleas,  553,  554 
by  flies,  552 
by  lice,  553 


Plague,  by  rats,  550,  551,  553 

varieties,  545 
Planococcus,  34 
Planosarcina,  34 
Plasmodium  falciparum,  482 

gametocytes  of,  483 
malariae,  471,  478 
gametocytes  of,  479 
meroblasts  of,  479 
spores  of,  477 
vivax,  471,  479 

developmental  cycle  of,  476 
gametocytes  of,  481 
Plasmolysis,  31 
Plate  cultures,  204 

disadvantages  of,  206 
leveling  apparatus  for  making,  205 
of  Koch,  201 

Platinum  needles  for  transferring  bac- 
teria, 202 
wires  for  bacteriologic  use,  202 

sterilization  of,  169,  202 
Pneumobacillus,  457.     See  also  Pneu- 

mococcus 

Pneumococcus,  444,  457 
cultivation,  458 
distribution,  457 
general  characteristics,  457 
Hiss'  inulin-serum-water  test  for  de- 
termining, 455 
morphology,  458 
pathogenesis,  459 
virulence,  461 
vital  resistance,  459 
Pneumonia,  444 

bacteriologic  diagnosis,  454 
broncho-,  461 
catarrhal,  461 
croupous,  444 
lobar,  444 
mixed,  461 
plague,  461 
sanitation  in,  456 
tuberculous,  461 
Poisoning,  cheese-,  247 
fish-,  248 

from  canned  goods,  248 
ice-cream,  247 
meat-,  247 
milk-,  247 
mussel-,  248 
Poisons,  food,  247 
Polar  granules  of  bacteria,  31 
Poliomyelitis,     acute     anterior,     381 . 
See  also  Acute  anterior  poliomyelitis 
Polyadenitis,  malignant,  543 .     See  also 

Plague 

Polyvalent  vaccines,  317 
Ponos,  530 

Post-mortems  on  animals,  228 
Potassium  permanganate  as  disinfect- 
ant, 178 

Potato,  cultures  on,  211 
Potato-cutter,  Ravenel,  198 
Potato- juice  as  culture-medium,  198 


Index 


801 


Potatoes  as  culture-medium,  197 
Pratt  and  Peabody's  method  of  isola- 
tion of  Bacillus  typhosus,  611 
Precipitate,  specific,  100 
Precipitation,  specific,  120 
Precipitinogen,  122 
Precipitins,  120,  122 
Predisposition,  84 
Prodigiosus  powder,  361 
Production    of    phosphorescence    by 

bacteria,  62 

Proskauer  and  Beck's  method  of  cul- 
tivation of  tubercle  bacillus,  667 
Protection  of  culture- media,  170 

of  instruments  and  glassware,  167 
Proteins,  defensive,  108 
Protista,  26 
Protophyta,  26 
Protozoa,  26,  44 

classification  of,  44,  45 

encystment  of,  49 

living,  observation  of,  161 

movement  of,  48 

nucleus  of,  48 

reproduction  of,  49 

size  of,  48 

staining,  162.     See  also  Staining 

structure  of,  46 
Pseudo-diphtheria  bacillus,  429.     See 

also  Bacillus  pseudo-diphtheria 
Pseudodysentery  bacillus,  648 
Pseudo-glanders  bacillus,  714 
Pseudo-influenza  bacillus,  466 
Pseudomonas,  35 
Pseudo-tetanus  bacillus,  351 
Pseudotuberculosis,  694 
Ptomains,  58 
Pulex  irritans,  553,  562 
Pure  cultures,  201,  208 
Pustule,  malignant,  359 
Putrefaction,  19,  58 
Putrefactive  ferment,  23 
Pyemia,  79 
Pyocyanase,  65,  323 
Pyocyanolysin,  323 

RABBIT  septicemia,  bacillus  of,  564 
Rabies,  363.  See  also  Hydrophobia 
Rats,  transmission  of  plague  by,  550, 

55i,  553 

Ravenel's  potato-cutter,  .198 

Ray-fungus,  732 

Reaction,  Calmette's  ophthalmo-tuber- 

culin,  679 

influences  on  growth  of  bacteria,  52 
Lignieres  cutituberculin,  679 
von  Pirquet's  dermotuberculin,  679 
Wassermann,  139 
Widal,  123 

Receptors,  112,  125 

Refined  tuberculin,  677 

Regressive  schizogony,  478 

Reichel  filter,  174 

Reichel  and  Engle's  method  of  stain- 
ing Negri  bodies,  369 


Relapsing  fever,  494 

bacteriologic  diagnosis,  501 
course,  500 

immunity  against,  501 
lesions,  501 

transmitted  by  ticks,  499 
vectors  of,  501 
Relative  immunity,  89 
Re"my's     method     of    cultivation    of 

Bacillus  typhosus,  606 
Reproduction  of  bacteria,  32 

of  protozoa,  49 
Respiratory       apparatus,       infection 

through,  74 
tract,  infection  with  tubercle  bacillus 

through,  669 

Retention  theory  of  immunity,  105 
Rhinoscleroma,  715 
Rhipicephalus  decoloratus,  494 
Rhizopoda,  45 
Rice-water      discharges      in      Asiatic 

cholera,  576 
Ringworm,  752 

Robinson's  method  of  disinfection,  182 
Romanowsky's    method    of    staining 

protozoa,  163 
Room,  disinfection  of,  182 
Ross'  method  of  staining  protozoa,  166 
Rossi's  method  of  staining  flagella,  160 
Rost's  method  of  cultivation  of  Bacil- 
lus leprae,  699 
Rothberger's  method  of  cultivation  of 

Bacillus  typhosus,  607 
Rotz,  706 

Roux's  bacteriologic  syringe,  222 
Roux  and  Nocard's  method  of  culti- 
vating tubercle  bacillus,  665 
Rubber  stoppers,  sterilization  of,  169 

SACCHAROMYCES  cerevisiae-,  39 
hominis,  39,  747 

Saccharomycosis  hominis,  747 

Sacs,  collodion,  preparation  of,  229 

Salamonsen's     method     of     making 
anaerobic  cultures,  220 

Salkowski's  test  for  indol,  62 

Salts  as  disinfectants,  177 

Sand  filter,   Petri's,   for  air-examina- 
tion, 236 

Sanitation  in  Asiatic  cholera,  580 
in  plague,  556 
in  pneumonia,  456 

Sapremia,  79 

Saprogens,  57 

Saprophytic  bacteria,  66 

Sarcina,  34 

Sarcopsylla  penetrans,  561 

Scarlatina,  streptococcus  in  blood  in, 

3i3 

Schaumorgane,  338 
Schering's  method  of  embedding,  150 
Schick's  reaction  in  diphtheria,  428 
Schizogony,  regressive,  478 
Schizonts,  475 
Schizotrypanum  cruzi,  518 


802 


Index 


Schizotrypanum  cruzi,  cultivation,  521 
morphology,  520 
pathogenesis,  521 
reproduction,  520 
transmission,  521 

Schlaffkrankheit,  506.  See  also  Sleep- 
ing sickness 

Schottelius  method  of  making  pure 
cultures  of  Spirillum  choleras  Asiat- 
icae,  572 

Schuffner's  granulations,  482 

Scissors,  sterilization  of,  169 

Scutulum,  755 

Sedgwick's    expanded    tube    for    air  - 

examination,  236 

method  of  quantitative  estimation 
of  bacteria,  235 

Seeding,  256 
tubes,  256 

Seitenkettentheorie  of  Ehrlich,  no 

Semmelformig,  394 

Sensitization  of  vaccines,  269 

Septic  tank  method  of  sewage  dis- 
posal, 57 

Septicemia,  79 

rabbit,  bacillus  of,  564 

Serum,  antipneumococcus,  456 
antirabic,  380 
antistreptococcus,  316 
antitubercle,  683 
anti  venomous,  132 
bacteriolytic,    therapeutic    uses    of, 

138 

Coley's,  316 
disease,  103 

in  testing  opsonic  value  of  blood,  273 
in  Wassermann  reaction,  281 
guinea-pig,  titration  of,  285 
hemolytic,  titration  of,  285 
treatment  of  Asiatic  cholera,  579 
Sewage,  colon  bacillus  in,  623 

disposal  by  septic  tank  method,  57 
Sexual  apparatus,  infection  with  tuber- 
cle bacillus  through,  670 
Shake  culture,  219 
Sheep    corpuscles,    titration    of,    for 

Wassermann  reaction,  285 
Shiga's  bacillus,  647.     See  also  Bacillus 

dysenteries 
Shiga-Kruse     variety     of     dysentery 

bacillus,  648 
Ship-fever,  540 
Siberian  pest,  352 
Sick-chambers,  disinfection  of,  175 
Sick-room,  disinfection  of  air  of,  176 
Silver  nitrate  as  disinfectant,  178 
Size  of  bacteria,  27     - 

of  protozoa,  48 
Skin,  bacteria  in,  68 

infection  through,  72 
Sleeping  sickness,  506 
clinical  picture,  506 
lesions  in,  516 
prophylaxis,  517 
specific  organism,  507 


Sleeping     sickness,     transmission     to 

lower  animals,  514 
Smallpox,  early  inoculation  for,  92 
Smith  fermentation-tube,  59 

method     of     determining     Bacillus 

coli  in  water,  249 
for  determining  nature  of  gases,  59 
modification  of  Newman's  method  of 

staining  flagella,  161 
of  Pitfield's  method  of  staining 

flagella,  159 
Smith's   (T.)    method  of    isolation  of 

tubercle  bacillus,  666 
Soil,  bacteria  in,  243 

FrankePs  method  of  estimating, 

243 

bacteriology  of,  243  . 
Solution,  Nessler's,  64 
Soor,  438 

Sore,  Oriental,  531,  533 
Sources  of  infection,  67 
Sozins,  108 
Species  of  bacteria,  identification  of, 

230 

Specific  action  of  toxins,  77 
affinity  of  cells  for  toxins,  78 
micro-organisms,  299 
Spermatoxin,  102 
Spermatozoits,  477 
Spinale  Kinderlahmung,  381 
Spirilla  resembling  cholera  spirillum, 

580 

table  for  differentiating,  588 
Spirillum-,  35 

choleras  Asiatics,  568 
cultivation,  572 
detection,  577 

Gordon's  method,  577 
Loffler's  method,  577 
distribution,  569 
general  characteristics,  568 
immunity  against,  578 
isolation,  572 
metabolic  products,  575 
morphology,  570 
pathogenesis,  575 
Schottelius'  method  of  making 

pure  cultures,  572 
specificity,  577 
staining,  571 
toxic  products,  575 
metchnikovi,  584.     See  also  Spiril- 
lum of  Gamaleia 
obermeieri,    494.     See    also    Spiro- 

chceta  obermeieri 
of  Finkler  and  Prior,  580 
cultivation,  580,  584 
metabolic  products,  582,  584 
morphology,  580,  583 
pathogenesis,  582,  584 
staining,  580 
of  Gamaleia,  584 
cultivation,  584 
immunity  against,  586 
metabolic  products,  585 


Index 


803 


Spirillum   of   Gamale'ia,    morphology, 

584 

pathogenesis,  585 

staining,  584 

vital  resistance,  585 
schuylkiliensis,  586 

cultivation,  586 

immunity  against,  587 

metabolic  products,  586 

morphology,  586 

pathogenesis,  587 

vital  resistance,  587 
Spirochasta,  36 
anserinum,  494 
berbera,  495 
carteri,  495 
duttoni,  494 
gallinarum,  494 
kochi,  495 
novyi,  495 
obermeieri,  494 

cultivation,  497 

general  characteristics,  496 

mode  of  infection,  498 

morphology,  496 

pathogenesis,  500 

staining,  497 
pallidum,  718.     See  also  Treponema 

pallidum 
persica,  495 
recurrentis,  494.  See  also  Spiro- 

chceta  obermeieri 
refringens,  718,  728 
theileri,  494 
vincenti,  433,  437 

Bacillus  fusiformis  and,  relation, 

434    . 

cultivation,  434 
morphology,  436 
Spiromonas,  36 
Spirosoma,  35 
Spirulina,  36 
Splenic  fever,  352 
Splenomegaly,    febrile    tropical,    525. 

See  also  Kala-azar 
Sporangia,  41 
Spores,  germination  of,  34 

staining,  156 
Sporocysts,  49 
Sporotrichosis,  759 
clinical  varieties,  764 
diagnosis,  agglutination  test,  764 
bacteriologic,  764 
complement-fixation  test,  764 
disseminated  gummatous,  764 

subcutaneous,  with  ulceration, 

764 

lesions,  763 
localized,  764 
mixed  forms,  764 
specific  organism,  759 
Sporotrichotic  chancre,  764 
Sporotrichum,  759 
beurmanni,  759 
asteroides,  759 


Sporotrichum  beurmanni,indicum,759 
gougerati,  759 
jeanselmei,  759 
schencki,  759 
cultivation,  761 
distribution  in  nature,  762 
lesions  caused  by,  763 
metabolic  products,  762 
morphology,  761 
pathogenesis,  762 
staining,  761 
vital  resistance,  762 
Sporozoa,  45 

furunculosa,  533 
Sporozoits,  475,  477 
Sporulation  of  bacteria,  32 
Spotted  fever,  386 
Sputum,  staining  tubercle  bacillus  in, 

659 

typhoid  bacilli  in,  598 
Stab  culture,  209 
Stain,  Mallory's  eosin-methylene  blue, 

155 
Staining  bacteria,  146 

aqueous  solutions  for,  148 
Gram's  method,  152,  153 

Nicolle's  modification,  154 
Gram-Weigert  method,  154 
Loffler's  method,  151 
Pfeiffer's  method,  151 
simple  method,  147,  150 
stock  solutions  for,  148 
Zieler's  method,  155 
flagella,  Loffler's  method,  158 

Newman's  method,  Smith's  modi- 
fication, 161 
Pitfield's  method,  159 

Smith's  modification,  159 
Rossi's  method,  160 
Van  Ermengem's  method,  159 
jar,  Coplin's,  151 

protozoa,  Biondi-Heidenhain  meth- 
od, 165 

cover-glasses  for,  162 
Heidenhain's  method,  165 
Marino's  method,  164 
Romanowsky's  method,  163 
Ross'  method,  166 
slides  for,  162 
tissue,  165 

Wright's  method,  163 
spores,  156 

Abbott's  method,  156 
Anjeszky's  method,  157 
Fiocca's  method,  157 
Moller's  method,  157 
Muir  and  Ritchie's  method,  156 
Standard  reaction  of  culture-media,  189 
Standardizing  freshly  isolated  cultures, 

214 

Staphylococci  of  man,  chief  types,  301 
Staphylococcus,  35 
citreus,  308 
epidermidis  albus,  300 
golden,  301 


804 


Index 


Staphylococcus  pyogenes  albus,  300  / 
aureus  et  albus,  302 
agglutination,  307 
bacterio-vaccination,  307 
colonies  of,  303 
cultivation,  303 
distribution,  302 
isolation,  303 
metabolic  products,  304 
morphology,  303 
pathogenesis,  306 
serum  therapy,  307 
staining,  303 
thermal  death  point,  304 
toxic  products,  305 
virulence,  307 
Staphylolysin,  305 

Starkey's  labyrinth,  Somers'  modifica- 
tion, 609 
method    of    isolation    of    Bacillus 

typhosus,  609 
Stegomyia  calopus,  537 

fasciata,  537 
Sterilization,  167 
by  filtration,  172 
in  autoclave,  171 
intermittent,  170 
methods  of,  169 
of  corks,  169 
of  culture-media,  170 
of  forceps,  169 
of  glassware,  167,  169 
of  instruments,  167 
of  knives,  169 
of  ligatures,  175 
of  platinum  wires,  169 
of  rubber  stoppers,  169 
of  scissors,  169 
of  surgical  instruments,  175 
of  wooden  apparatus,  169 
Sterilizer,  Arnold's  steam,  171 

hot-air,  169 
Sternberg's  bulbs,  250 

method    of   determining  germicidal 

value,  253 
Stern's  method  of  staining  Treponema 

pallidum,  720 

Stewart  cover-glass  forceps,  149 
Stock  vaccines,  265 
Stomach,  bacteria  in,  70 
Stomatitis,  parasite,  438 
Straus'  method  of  diagnosis  of  glanders, 

709 

Streptobacillus,  403 
Streptococcus,  35 
brevis,  310 
conglo  meratus,  311 

diffusus,  311 
erysipela'tis,  318 
in  blood  in  scarlatina,  313 
mucosus,  317 
pyogenes,  308 
cultivation,  310 
differential  features,  311 
general  characteristics,  308 


Streptococcus  pyogenes,  isolation,  310 
metabolic  products,  314 
morphology,  309 
pathogenesis,  312 
staining,  310 
toxic  products,  315 
virulence  of,  313 
'  vital  resistance  of,  311 
vaccine,  317 
viridans,  312 
Streptokolysin,  315 
Streptothrix,  37 
actinomyces,  37 
farcinica,  37 
madurae,  37 
Structure  of  bacteria,  30 

of  protozoa,  46 

Subculture  tubes,  block  for,  257 
Subinfection,  66 
Substance-sensibilisatrice,  118 
Sucholotoxin,  627 
Sugar  bouillon,  192 
Sulphur  grain,  734 
Suppuration,  299 

bacteria  associated  with,  299 
Surgical  contributions,  20 

instruments,  sterilization  of,  175 
Susceptibility  from  diet,  85 
from  exposure,  85 
from  fatigue,  85 

from  inhalation  of  noxious  vapors,  84 
from  intoxication,  85 
from  morbid  conditions  in  general,  86 
from  mutilation  of  body,  86 
from  traumatic  injury,  86 
of  host,  84 
Susotoxin,  627 
Suspension,  bacterial,  270 
Sutures,  disinfection  of,  172 
Swarmers,  655 

Swine-plague,  bacillus  of,  566 
Symbiosis,    influences    on    growth    of 

bacteria,  54 
Synopsis     of     groups     of      bacteria, 

Chester's,  231-233 
Syphilis,  718 

bacillus  of,  718.     See  also  Treponema 

pallidum 
diagnosis,  726 

by  Noguchi's  luetin  reaction,  727 
by  serum,  726 

by  von  Pirquet  tuberculin  reac- 
tion, 726 

by  Wassermann  reaction,  279,  726 
lesions,  726 
Syphilitic  antigen,  279 
Syringe,  Altmann's,  223 
Koch's,  222 
Meyer's,  222 
Roux's  bacteriologic,  222 
System  hemolytic,  286 

TABARDILLO,  540 

Temperature,  influences  on  growth  of 
bacteria,  55 


Index 


805 


Temperature,   sensitivity  of   tubercle 

bacillus  to,  669 
Terminal  infections,  312 
Test,    tuberculin,    for    bovine    tuber- 
culosis, 689 

Tetanolysin,  76,  102,  131,  345,  347 
Tetanospasmin,  76,  131,  345 
Tetanus,  340 
antitoxin,  131 
ascendens,  346 
bacillus  of,  340.     See   also   Bacillus 

tetani 

clonic  convulsions  in,  346 
descendens,  346 
dolorosus,  347 
lockjaw  of,  347 
opisthotonos  of,  347 
pathogenesis,  347 
prophylactic  treatment,  351 
tonic  convulsions  in,  346 
trismus  of,  347 
Tetracoccus,  34 

Theobald  Smith  phenomenon,  103 
Therapy,  blood-serum,  24,  222 
Thermal  death-point  of  bacteria,  de- 
termination of,  249 
Thermophilic  bacteria,  55 
Thrush,  438 
Tick  fever,  494 
Ticks,  501 

in  transmission   of   relapsing   fever, 

500 

Tinea  circmata,  752 
favosa,  755 
imbricata,  752 
trichophytina,  752 
unguium,  752 
versicolor,  752 
Toxemia,  79 
Toxins,  extracellular,  76 
i  ntracellular,  75 
specific  action  of,  77 

affinity  of  cells  for,  78 
Toxoids,  in 
Toxophylaxins,  108 
Toxosozins,  108 
Trachea,  bacteria  in,  72 
Treponenia,  36 
pallidulum,  729 
pallidum,  718 
cultivation,  722 

Noguchi's  method,  722 
distribution,  722 
film  staining,  719 

Ghoreyeb's  method,  719 
Goldhorn's  method,  719 
Stern's  method,  720 
general  characteristics,  718 
identifying  by  Bum's  India  ink 

method,  721 
morphology,  718 
pathogenesis,  725 
section  staining,  721 

Levaditi's  method,  721 
Manouelian's  method,  722 


Treponema  pallidum,  specificity,  725 
pertenue,  729 

cultivation,  730 

morphology,  730 

pathogenesis,  730 

staining,  730 

Trichomonas  intestinalis,  655 
Trichophyton,  41 
acuminatum,  752 
circonvulatum,  752 
crateriforme,  752 
effractum,  752 
exsiccatum,  752 
flavum,  752 
fulmatum,  752 
glabrum,  752 
megalosporon,  752 
microsporon,  752 
pilosum,  752 
plicatili,  752 
polygonum,  752 
regulare,  752 
sulphureum,  752 
tonsurans,  752 

cultivation,  753 

morphology,  753 

pathogenesis,  754 
umbilicatum,  752 
violaceum,  752 
Trikresol,  179 
Trismus,  347 
Tropical  ulcer,  531 

organism,  533 

preventive  inoculation,  534 

transmission,  534 

treatment,  534 
Trypanosoma  avium,  508 
brucei,  508 


rr    520.     See     also     Schizotry- 

panum  cruzi 
equinum,  508 
equiperdum,  514 
gambiense,  506,  508 

cultivation,  510 

distribution  in  body,  515 

morphology,  509 

pathogenesis,  514 

reproduction,  510 

staining,  510 

transmission,  511 
granulosum,  508 
lewisi,  508 


rhodesiensi,  506.     See  also  Trypano- 

soma gambiense 
rotatOflUm,  508 
jjolete,  508 
theileri,  508 
trajnsvaliense,  508 


various  species,  508 
Trypanosomiasis,  American,  518 
human,     506.     See     also     Sleeping 
sickness 


8o6 


Index 


Tsetse  fly,  517 

appearance,  517 
breeding  habits,  518 
disease,  512 
habitat,  517 
habits,  517 
larva  of,  518 

table  for  identification  of,  518 
Tube,  expanded,  Sedgwick  and  Tuck- 
er's, for  air-examination,  236 
Keidel,  281 

Tubercle  bacillus,  656.     See  also  Bacil- 
lus tuberculosis 
Tubercles,  673 
crude,  674 
giant-cells  in,  672 
healed,  675 
miliary,  673 
of  Babes,  372 

Tuberculin,  concentrated,  677 
crude,  677 
dangers  from,  678 
Denys',  680 

effect  on  tubercle  bacillus,  678 
Koch's,  676 
preparation,  678 
refined,  677 

test  for  bovine  tuberculosis,  689 
Tuberculin-R,  680 
Tuberculin-TR,  680 
Tuberculinic  acid,  676 
Tuberculocidin,  680 
Tuberculosamin,  676 
Tuberculosis,  656 

bacillus  of,  656.     See  also  Bacillus 

tuberculosis 
bovine,  685 

communicability  to  man,  686 
lesions,  686 
prophylaxis,  688 
tuberculin  test  for,  689 
diagnosis,     Calmette's    ophthalmo- 

tuberculin  reaction,  679 
Morro's  method,  679 
von  Pirquet's  cutaneous  method, 

^679 
Ligniere's    modification, 

679 

Wolff-Eisner   oph thai  mo-tubercu- 
lin method,  680 
distribution,  656 
fish,  691 
fowl,  690 
latent,  674 
lesions,  671 

prophylaxis  against,  684 
pseudo-,  694 
specific  organism,  656 
Tuberculous  pneumonia,  461 
Tubes,  Esmarch,  207* 

seeding,  256 

Tucker's     expanded     tube     for     air- 
examination,  236 
Typhoid  carriers,  595 
fever,  589 


Typhoid  fever,  bacillus  of,  589 

bacteriologic  diagnosis,  603 

blood-culture  in,  603 

conjunctival  reaction  in,  604 

jiistologic  lesions,  597 

i  n  lower  animals,  599 

isolation  of  bacillus  from  feces,  604 

pathogenesis,  597 

prophylactic  vaccination  against, 
600 

prophylaxis,  599 

specific  therapy,  601 

Widal  reaction  in,  603 
pig,  625 
Typhus   abdominalis,   540,   589.     See 

also  Typhoid  fever 
exanthematicus,  540 
fever,  540 

inoculation  into  animals,  541 

transmission  by  lice,  542 
Tyrotoxicon,  58,  247 
Tyrotoxism,  247 

ULCER,  tropical,  531 
Umstimmung,  727 

Unit,  amboceptor,  in  Wassermann  re- 
action, 286 

hemolytic,  286 
Urethra,  bacteria  in,  71 
Urine,  staining  smegma  bacillus  in,  663 
tubercle  bacillus  in,  662 

typhoid  bacilli  in,  598 
Uterus,  bacteria  in,  71 

VACCINATION  against  typhoid,  600 

efficient,  94 

inefficient,  94 

Jennerian,  93 

Pasteur,  95 
Vaccines,  93 

autogenous,  265 

bacterio-,  263 

dosage  of,  268 

method  of  making,  265 

polyvalent,  317 

sensitization  of,  269 

stock,  265 

streptococcus,  317 
Vagina,  bacteria  in,  71 
Van  Ermengem's  method  of  staining 

flagella,  159 

Vectors  of  relapsing  fever,  501 
Vegetables,  bacteria  in,  243 

Bacillus  typhosus  in,  595 
Vibrio,  35 

lineola,  728 

proteus,  580.     See    also    Spirillum, 

Finkler  and  Prior 
Vibrion  septique,  329 
Vibrionensepticaemia,  586 
Vincent's  angina,  433 
Virulence,  79 

decrease  of,  80 

increase  of,  80 


Index 


807 


Virulence,  increase  of,  by  addition  of 
animal  fluids  to  culture-media, 
81 

by  passage  through  animals,  80 
by  use  of  collodion  sacs,  81 
Viruses,  filterable,  28 

invisible,  28 
Von   Pirquet   cutaneous   reaction  for 

diagnosis  of  syphilis,  726 
dermotuberculin  reaction,  679 

WASSERMANN'S  method  of  cultivation 

of  Micrococcus  gonorrhceae,  396 
reaction,  139,  279,  726 
amboceptor  dose  in,  286 

unit  in,  286 
antigen  in,  279 
blood-corpuscles  for,  283 
complement  for,  282 
fixation  test  of,  291 
hemolytic  amboceptor  for,  284 
titration  of,  284 

system  in,  286 

test  of,  291 
nature  of,  294 

Noguchi's  modification  of,  294 
obtaining  blood  for,  282 
reagents  employed,  279 
serum  to  be  tested  in,  281 
technic  of,  279,  280 
theoretic  basis  of,  280 
titration  of  antigen  in,  288 

of  guinea-pig  serum  for,  285 

of  hemolytic  serum  for,  285 

of  sheep  corpuscles  for,  285 
validity  of,  294 
Water,  Bacillus  coli  in,  230 

Smith's  method  of  determining, 

239 

bacteria  in,  50,  237 
number  of,  238 

method  of  determining,  237 
bacteriology  of,  237 
drinking,  colon  bacillus  in,  622 
Water-bath,  254,  255 
Welch's    method    of    staining    Diplo- 

coccus  pneumonia?,  445 
Wertheim's  method  of  cultivation  of 

Micrococcus  gonorrhceae,  395 
Whey,  Petruschkey's,  199 
Whooping-cough,  441 
Widal  reaction,  123 

in  typhoid  fever,  603 


Wildseuche,  bacillus  of,  566 
Willcomb's  method  of   counting   bac- 
teria, 238 
Williams    and   Lowden's    method    of 

staining  Negri  bodies,  368 
Winslow's  method  of  counting  bacteria, 

238 
Wires,  platinum,  for  bacteriologic  use, 

202 

Wolfhugel  apparatus  for  counting  col- 
onies of  bacteria  on  plates,  237 
Wooden  apparatus,  sterilization  of,  169 

tongue,  732 

Wool-sorters'  disease,  358 
Wounds,  disinfection  of,  175 

tubercle  bacillus,  infection  through, 

671 

Wright's  blood-stain  for  protozoa,  163 
method  of  counting  bacteria  in  sus- 
pension, 238 
of  making  anaerobic  cultures,  218, 

220 

Wiirtz  and  Kashida's  method  of  culti- 
vation of  Bacillus  typhosus,  606 

XENOPSYLLA  cheopis,  552,  554,  561,562 
Xerosis,  bacillus  of,  431 
X-rays,  influences  on  growth  of  bac- 
teria, 53 

Y  BACILLUS,  648 

Yaws,      729.       See     also     Frambesia 
tropica 

Yeasts,  38 

Yellow  fever,  536 

mosquitoes  and,  537 

prophylaxis,  539 

rules  for  prevention,  538 

Young's    method    of    cultivation    of 
Micrococcus  gonorrhceae,  395 

ZENKER'S  fluid,  149 

Ziehl's    method    of    staining    Bacillus 

typhosus,  590 
tubercle  bacillus,  66 1 
Zieler's  method  of  staining,  155 
Zinsser's  method  of  making  anaerobic 

cultures,  218 

Zopf's  Bacterium  pneumonias,  457 
Zur  Nedden's  bacillus,  409 
Zygospores,  41 
Zygote,  477 
Zymogens,  57 


* 


TC  0047V 


33  06 


MS 


BIOLOGY         , 
LIBRARY        ;^ 

UNIVERSITY  OF  CALIFORNIA  LIBRARY 


